EP2012799A2 - Metabolically stable alkoxyalkyl esters of antiviral or antiproliferative phosphonates, nucleoside phosphonates and nucleoside phosphates - Google Patents

Metabolically stable alkoxyalkyl esters of antiviral or antiproliferative phosphonates, nucleoside phosphonates and nucleoside phosphates

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Publication number
EP2012799A2
EP2012799A2 EP07760797A EP07760797A EP2012799A2 EP 2012799 A2 EP2012799 A2 EP 2012799A2 EP 07760797 A EP07760797 A EP 07760797A EP 07760797 A EP07760797 A EP 07760797A EP 2012799 A2 EP2012799 A2 EP 2012799A2
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EP
European Patent Office
Prior art keywords
group
propyl
cidofovir
phosphonate
nucleoside
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP07760797A
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German (de)
French (fr)
Other versions
EP2012799A4 (en
EP2012799B1 (en
Inventor
Karl Y. Hostetler
James R. Beadle
Jacqueline Ruiz
Merrick R. Almond
George R. Painter
Timothy A. Riley
Paula Francom
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University of California
Chimerix Corp
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University of California
Chimerix Corp
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Publication of EP2012799A2 publication Critical patent/EP2012799A2/en
Publication of EP2012799A4 publication Critical patent/EP2012799A4/en
Application granted granted Critical
Publication of EP2012799B1 publication Critical patent/EP2012799B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/657163—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom
    • C07F9/657172—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms the ring phosphorus atom being bound to at least one carbon atom the ring phosphorus atom and one oxygen atom being part of a (thio)phosphinic acid ester: (X = O, S)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs

Definitions

  • the present invention relates to orally administered drugs for treatment of viral infections and certain cancers.
  • the present invention relates to metabolically stable alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates, compositions containing them, processes for obtaining them, and their use in treating a variety of medical disorders, in particular viral infections, cancers and the like.
  • Nucleoside phosphonates have antiviral, antiproliferative and a variety of other therapeutic benefits.
  • antiviral nucleoside phosphonates such as, for example, cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like, as well as the 5'-phosphonates and methylene phosphonates of azidothymidine (AZT), ganciclovir, acyclovir, and the like.
  • the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond.
  • a methylene group replaces the 5'- hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.
  • nucleoside phosphonate diphosphate which represents the equivalent of nucleoside triphosphates.
  • Antiviral nucleoside phosphonate diphosphates are selective inhibitors of viral RNA or DNA polymerases or reverse transcriptases. That is to say, their inhibitory action on viral polymerases is much greater than their degree of inhibition of mammalian cell DNA polymerases ⁇ , ⁇ and ⁇ or mammalian RNA polymerases.
  • antiproliferative nucleoside phosphonate diphosphates inhibit cancer cell DNA and RNA polymerases and may show much lower selectivity versus normal cellular DNA and RNA polymerases.
  • antiviral and antiproliferative compounds are the antiviral nucleoside phosphonates.
  • Two representative structures of this class of compounds, namely CDV and HPMPA, are set forth below:
  • Another class of phosphonates is the 5'-phosphonates and methylene phosphonates of azidothymidine, ganciclovir, acyclovir, and the like.
  • the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides (ganciclovir, penciclovir, acyclovir), which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond.
  • a methylene group replaces the 5'-hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.
  • nucleoside phosphates such as, acyclovir monophosphate, 2'-O-methyl-guanosine-5'-phosphate, 2 '-O-methyl-cytidine-5 '-phosphate and 2 '-C-methyl-cytidine-5 '-phosphate.
  • nucleoside phosphates such as, acyclovir monophosphate, 2'-O-methyl-guanosine-5'-phosphate, 2 '-O-methyl-cytidine-5 '-phosphate and 2 '-C-methyl-cytidine-5 '-phosphate.
  • Various substituent groups may be attached to phosphonates and phosphates to produce derivatives having various degrees of pharmacological potency.
  • One class of derivative compounds are the alkoxyalkyl esters, such as hexadecyloxypropyl cidofovir (HDP-CDV), which is illustrated by the following general structure:
  • HDP-CDV Hexadecyloxypropyl cidof ovir
  • CDV itself is not orally active; however esterification of CDV with certain alkoxyalkanols such as hexadecyloxypropanol dramatically increases its antiviral activity and selectivity in vitro and confers a degree of oral bioavailability.
  • alkyl chain length of these CDV analogs is related to solubility and the ability of the compounds to associate with biomembranes.
  • alkoxyalkyl esters of nucleoside phosphates and phosphonates such as hexadecyloxypropyl-cidofovir (HDP-CDV)
  • HDP-CDV hexadecyloxypropyl-cidofovir
  • Orally administered drugs are usually taken up from the small intestine into the portal vein, which exposes the drug to potentially rapid lipid metabolism in the enterocytes of the small intestines and in the liver.
  • Alkoxyalkyl esters of phosphates and phosphonates can be incorporated into cell membranes where the phosphate or phosphonate is subsequently liberated inside the cell or can be oxidatively metabolized by the cytochrome P450s such as CYP3A4 in the liver or intestine leading to omega oxidation of the alkyl chain followed by beta oxidation. It has recently been determined that alkoxyalkyl esters of phosphates and phosphonates can be oxidized at the terminal end of the alkyl chain by omega oxidation and are further degraded by beta oxidation to short chain inactive metabolites.
  • nucleoside monophosphonate HDP-CDV may be very rapid and is deleterious to the intended pharmacologic effect of the compounds.
  • the inactive metabolite is water soluble, virologically inactive, and rapidly excreted in the urine. Rapid metabolism by this pathway may lower plasma levels of the prodrug, and reduce the antiviral efficacy of HDP-CDV and alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates.
  • the present invention includes esters of phosphonates, nucleoside phosphonates and nucleoside phosphates (referred to collectively hereinafter as esters) that are resistant to metabolic inactivation resulting from oxidation of these compounds in the liver. More specifically, the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted alkoxyalkyl esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing metabolic stability during absorption in the small intestine, first pass liver metabolism and subsequent distribution to peripheral tissues. Included in the present invention are methods for using said esters for treating various diseases and conditions.
  • the compounds and methods of this invention are based upon the unique insight that ⁇ -oxidation of lipid esters of phosphonates and phosphates may be slowed by placing a blocking group or groups at or near the penultimate carbon of the alkyl chain.
  • Potential blocking groups include, but are not limited to alkyl groups, including, but not limited to methyl, ethyl and propyl, cyclopropyl and halogens.
  • Potential blocking groups also include alkenyl groups containing one or more double bonds, including a terminal double bond.
  • Phosphonate compounds contemplated for use in accordance with the present invention include those having antiviral and antiproliferative activity.
  • nucleoside analogs with antiviral activity against hepatitis C which can be converted to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates.
  • nucleosides in this class of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine.
  • Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates are set forth in the references cited in Table 2.
  • nucleoside analogs with antiviral activity against hepatitis B which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates.
  • exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various ⁇ -L-2'- deoxycytidine, ⁇ -L-2'-deoxyadenine and ⁇ -L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45[l):229-235).
  • Anticancer agents may also be derivatized according to the method of this invention.
  • Some subject compounds include but are not limited to (E)-2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- ⁇ -D- arabinosyl)cytosine (4'-thio-FAC).
  • nucleosides may also become active when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol.
  • Non- nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl groups of the invention including, but not limited to topotecan by phosphorylating and esterifying an available hydroxyl group.
  • Etoposide may be derivatized by attaching metabolically stable groups of the invention to the phosphate residue of etoposide.
  • Phosphonate and phosphate analogs contemplated for use in accordance with the present invention are selected to improve the bioactivity, selectivity, and/or bioavailability of the antiviral or antiproliferative compounds.
  • compositions containing the analogs of the phosphonate compounds described herein there are provided pharmaceutical formulations containing the analogs of the phosphonate compounds described herein.
  • therapeutic methods e.g. methods for treating viral infections and methods for treating disorders caused by inappropriate cell proliferation, e.g. cancer and the like.
  • Figure 1 illustrates schematically antiviral activation and metabolic inactivation pathways for hexadecyloxypropyl cidofovir (HDP-CDV).
  • Figure 2 depicts representative structures of "Metabolism Resistant" lipophilic esters of cidofovir.
  • Figure 3 depicts a graph of the % of drug remaining versus time for HDP-
  • CDV and 15-methyl-HDP-CDV 15M-HDP-CDV. This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative
  • the present invention includes chemical methods for synthesizing alkoxyalkyl esters having certain moieties at or near the omega end of the alkyl chain which block or slow degradation and metabolic inactivation.
  • the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing resistance to oxidation.
  • Phosphonates, nucleoside phosphonates and nucleoside phosphates having antiviral or anticancer activity are subjects of the invention.
  • a or “an” entity refers to one or more of that entity; for example, a phosphonate refers to one or more phosphonates.
  • a phosphonate refers to one or more phosphonates.
  • the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein.
  • the method of the invention is not limited to any particular isomer and can be extended to the S enantiomer, the R enantiomer or racemic mixtures thereof.
  • prodrug refers to derivatives of pharmaceutically active compounds that have chemically or metabolically cleavable groups and become the pharmaceutically active compound by solvolysis or under in vivo physiological conditions.
  • purine or pyrimidine base includes, but is not limited to, 6- alkylpurine and N 6 -alkylpurines, N 6 -acylpurines, N 6 -benzylpurine, 6-halopurine, N 6 - acetylenic purine, N -acyl purine, N -hydroxyalkyl purine, 6-thioalkyl purine, N - alkylpurines, 7-deazapurines, N 4 -alkylpyrimidines, N 4 -acylpyrimidines, A- halopyrimidines, N 4 -acetylenic pyrimidines, 4-amino and N 4 -acyl pyrimidines, A- hydroxyalkyl pyrimidines, 4-thioalkyl pyrimidines, thymine, cytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil, C -alkylpurines, N 6
  • ring- expanded and open-ring cogeners of any of the aforementioned purines.
  • Functional oxygen and nitrogen groups on the base can be protected as necessary or desired.
  • Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p- toluenesulfonyl.
  • Preferred bases include cytosine, 5-fluorocytosine, uracil, thymine, adenine, guanine, xanthine, 2,6-diaminopurine, 6-aminopurine, 6-chloropurine and 2,6- dichloropurine.
  • alkyl refers to a saturated straight or branched hydrocarbon.
  • the alkyl group can be optionally substituted with one or more halogens selected from the group consisting of F, Cl, Br or I.
  • alkenyl refers to a partially unsaturated straight or branched hydrocarbon.
  • the alkenyl group can be optionally substituted with one or more halogens selected from the group consisting of
  • protected refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes.
  • oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis. Suitable protecting groups are described, for example, in Greene, et al., "Protective Groups in Organic Synthesis,"
  • nucleoside phosphonates of the instant invention can be generally represented by the following structures.
  • R is selected from the group consisting of -R 1 -O-R 2 , wherein R 1 is selected from the group consisting of an optionally substituted C 1 to C 11 alkyl group and R 2 is selected from the group consisting of a C 6 to Cn alkyl group or a C 6 to Cn alkenyl group; wherein said Ce to Cn alkyl group is substituted with one or more alkyl groups selected from the group including, but not limited to methyl, ethyl, propyl, or cycloalkyl, including, but not limited to cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said C 6 to C 17 alkyl group includes one or more substituents at or near the terminal position of the alkyl group, in particular at the terminal or penultimate position; and wherein said Ce to Cn alkenyl group is optionally-substituted with an alkyl group selected from the group including, but
  • B is selected from a purine or pyrimidine base
  • A is a counterion selected from the group including, but not limited to H + , Li + , Na + , K + ,
  • R is selected from the group of compounds having the general structure:
  • R is selected from the group of compounds having the general structure:
  • R is selected from the group of compounds having the general structure:
  • X is a halogen.
  • X is F.
  • R is selected from the group consisting of one of the structures set forth in Figure 2.
  • derivatized nucleoside phosphonates are analogs of cyclic cidofovir or cidofovir which can be generally represented by the following structures:
  • the derivatized nucleoside phosphonates are analogs of 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)-adenine ((S)-HMPMA) which can be generally represented by the following structure:
  • cyclic cidofovir, cidofovir and HPMPA included in the present invention include the following compounds: 3-(12-methyltridecyloxy)propyl cyclic cidofovir, 3-(13-methyltetradecyloxy)propyl cyclic cidofovir, 3-(14- methylpentadecyloxy)propyl cyclic cidofovir, 2-(17-methyloctadecyloxy)ethyl cyclic cidofovir, 3-(15-methylhexadecyloxy)propyl cyclic cidofovir, 3-(15- methylhexadecyloxy)ethyl (S)-cyclic HPMPA, 3-(15-methylhexadecyloxy)propyl (S)- cyclic HPMPA, 2-(17-methyloctadecyloxy)ethyl-(S)-cyclic HPMPA, 3-(3(12-methyltride
  • nucleoside phosphates and analogs thereof of the instant invention can be generally represented by the following structures.
  • R is an alkoxyalkyl group having a structure as defined above and B is a substituted or unsubstituted pyrimidine base or their open ring congeners.
  • Representative examples of nucleosides in this group of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine.
  • Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'- phosphates or their alkoxyalkylglycerol phosphates are set forth in the references cited in Table 2.
  • nucleoside analogs with antiviral activity against hepatitis B which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates.
  • exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various ⁇ -L-2'- deoxycytidine, ⁇ -L-2'-deoxyadenine and ⁇ -L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45£l):229-235).
  • Phosphates of non-nucleoside antivirals are also subjects of the invention including, but not limited to, zanamivir (Relenza ® ).
  • Anticancer agents may also be derivatized according to the method of this invention.
  • Some representative compounds include, but are not limited to 2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- ⁇ -D- arabinosyl)cytosine (4'-thio-FAC).
  • nucleosides may also become more metabolically stable when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol.
  • Non-nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl esters of the invention including, but not limited to topotecan by coupling to an available hydroxyl group.
  • Etoposide may be coupled to the metabolically stable alkoxyalkyl esters of the invention by attachment to the phosphate residue of etoposide.
  • Tables 1 and 2 provide examples of compounds, which may be subjected to the chemical steps of the invention.
  • the references cited in these Tables are hereby incorporated by reference in their entirety.
  • Table 1. References citing illustrative phosphonates, nucleoside phosphonates and nucleoside phosphates for use according to the method of this invention
  • compositions can be prepared in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions, or together with carriers for topical applications.
  • Pharmaceutical formulations containing compounds of this invention can be prepared by conventional techniques, e.g., as described in Remington's Pharmaceutical Sciences, 1985.
  • the pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier.
  • solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or distearate, alone or mixed with a wax.
  • a solid carrier is used for oral administration, the preparation may be tabletted or placed in a hard gelatin capsule in powder or pellet form.
  • the amount of solid carrier will vary widely, but will usually be from about 25 mg to about I g.
  • a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
  • Tablets are prepared by mixing the active ingredient (that is, one or more compounds of the invention), with pharmaceutically inert, inorganic or organic carrier, diluents, and/or excipients.
  • excipients which can be used for tablets are lactose, maize, starch or derivatives thereof, talc, stearic acid or salts thereof.
  • suitable excipients for gelatin capsules are vegetable oils, waxes, fats, semisolid, and liquid polyols.
  • the preparation may contain a compound of the invention dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application.
  • a liquid carrier in particular, an aqueous carrier
  • the carrier may contain solubilizing agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.
  • compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • Suitable excipients for the preparation of solutions and syrups are water, polyols, sucrose, invert sugar, glucose, and the like.
  • Suitable excipients for the preparation of injectable solutions are water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • the pharmaceutical products can additionally contain any of a variety of added components, such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, antioxidants, diluents, and the like.
  • the pharmaceutical compositions of the invention may comprise a compound according to the general formula combined with one or more compounds exhibiting a different activity, for example, an antibiotic or other pharmacologically active material. Such combinations are within the scope of the invention.
  • This invention provides methods of treating disorders related to viral infections, inappropriate cell proliferation, and the like. The methods particularly comprise administering to a host in need thereof a therapeutically effective amount of the prodrugs of this invention. Thus, in one aspect of the invention there are provided methods for treating disorders caused by viral infections.
  • Indications appropriate to such treatment include susceptible viruses including, but are not limited to human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), human herpes virus 6 and 8, cytomegalovirus (CMV), hepatitis B and C virus, Epstein-Barr virus (EBV), varicella zoster virus, and diseases caused by orthopox viruses (e.g., variola major and minor, vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebola virus, papilloma virus, and the like, lymphomas, hematological disorders such as leukemia, and the like, and cancers caused by viruses such as cervical cancer which is caused, in most cases, by the high risk subtypes of human papilloma virus.
  • HCV human immunodeficiency virus
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • cancers such as melanoma, lung cancers, pancreatic cancer, stomach, colon and rectal cancers, prostate and breast cancer, the leukemias and lymphomas, and the like.
  • Anti-cancer compounds which can be converted to their nucleotide phosphonates or nucleoside-5 '-phosphates for use as compounds of this invention include, but are not limited to, cytarabine (ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine, decitabine, cladribine, fludarabine, pentostatin (2'-deoxycoformycin), 6-mercaptopurine and 6-thioguanine and substituted or unsubstituted ara-adenosine (ara-A), ara-guanosine (ara-G), and ara- uridine (ara-U).
  • Anticancer compounds of the invention may be used alone or in combination with other antimetabolites or with other classes of anticancer drugs such as alkaloids, topoisomerase inhibitors, alkylating agents, antitumor antibiotics, and the like.
  • the prodrugs of the invention can be administered orally, parenterally, topically, rectally, and through other routes, with appropriate dosage units, as desired.
  • parenteral refers to subcutaneous, intravenous, intra-arterial, intramuscular or intravitaeal injection, or infusion techniques.
  • topically encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and mucous membranes of the mouth and nose and in toothpaste.
  • “Therapeutic” as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals.
  • “Pharmaceutically or therapeutically effective dose or amount” refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired.
  • a "host” or “patient” is a living subject, human or animal, into which the compositions described herein are administered.
  • the "effective amount" is determined with reference to the recommended dosages of the antiviral or anticancer parent compound.
  • the selected dosage will vary depending on the activity of the selected compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors, including the body weight, general health, diet, time, and route of administration and combination with other drugs, and the severity of the disease being treated.
  • the compounds of the present invention are dispensed in unit dosage form comprising 1% to 100% of active ingredient.
  • the range of therapeutic dosage is from about 0.01 to about 1,000 mg/kg/day with from about 0.10 mg/kg/day to 100 mg/kg/day being preferred, when administered to patients, e.g., humans, as a drug.
  • Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient.
  • Compounds of the invention can be prepared in a variety of ways, as generally depicted in Schemes 3 to 7 in Examples 1-9.
  • Example 1 (Scheme 1) outlines a general method for the synthesis of branched alkoxyalkanols having the general formula:
  • Example 2 (Scheme 2) outlines a general method for the synthesis of branched methylalkoxyalkyl esters from cyclic phosphonates. Cyclic cidofovir was used in this Example for purposes of illustration, however this method can be extended to the use of virtually any cyclic phosphonate of interest.
  • Examples 3 and 4 describe the synthesis of two specific branched alkoxyalkyl esters, namely 3-(phytanyloxy)propyl cidofovir and 15-methylhexadecyloxypropyl cidofovir (15-Me HDP-CDV), ammonium, using slight variations of the methods described in Examples 1 and 2.
  • Example 5 describes a general method for the synthesis of the branched methylalkoxyalkyl esters of the instant invention from p- toluenesulfonyloxymethyl phosphonates.
  • Example 6 (Scheme 5) outlines a general method for the synthesis of alkenyloxyalkyl esters having a terminal double bond. The nucleoside phosphonate cidofovir was used for purposes of illustration resulting in the synthesis of compound 26, hexadec-15-enyl-oxypropyl-cidofovir.
  • Examples 7-9 outline general methods for the synthesis of various halogenated alkoxyalkyl esters using CDV and HPMPA for purposes of illustration. Synthesis of the following compounds are exemplified: 3-(15- fluorohexadecyloxy)propyl cidofovir, 3-(15-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(15-fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(15- fluorohexadecyloxy)propyl-(S)-cyclic HPMPA, 3-(16-fluorohexadecyloxy)propyl cidofovir, 3-(16-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(16- fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(16-fluorohexadecy
  • Examples 10-12 illustrate the antiviral activity representative penultimate branched methyl alkoxyalkyl esters of CDV and HPMPA.
  • the results are set forth in Tables 3-6.
  • penultimate branched chain analogs of (S)-HPMPA were highly active against vaccinia and cowpox in vitro and penultimate branched chain alkoxyalkyl cidofovir esters were effective against ectromelia virus in vitro at submicromolar EC50s.
  • the branch chain analogs of (S)-HPMPA and CDV were all fully protective against death from lethal poxvirus infection at doses of 5 mg/kg/day or greater. 15M-HDP-(S)-HPMPA appeared to be more active than HDP-(S)-HPMPA.
  • FIG. 13 The stability of the compounds of the invention is illustrated in Example 13, the data for which is set forth in Figure 3.
  • Figure 3 depicts a graph of the % of drug remaining versus time for HDP-CDV and 15-methyl-HDP-CDV (15M-HDP-CDV) after incubation for various times as indicated in the presence of liver S 9 preparations. This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver and human liver S 9 fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative HDP-CDV.
  • Reagents a) magnesium, THF; b) Li 2 CuCl 4 , THF; c) methanesulfonyl chloride, triethylamine, CH 2 Cl 2 ; d) 1,2-ethanediol or 1,3-propanediol, NaH, N 5 N-DMF
  • 12-methyltridecan-l-ol was prepared from 9-bromononan-l-ol and 3- methylbutylbromide in 51% yield.
  • the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal 19:199-204).
  • 13-methyltetradecan-l-ol was prepared from 10-bromodecanol and 3- methylbutylbromide in 62% yield.
  • the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
  • 14-methylpentadecan-l-ol was prepared from 9-bromononanol and 5- methylhexylbromide in 55% yield.
  • the 1 H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
  • 15-methylhexadecan-l-ol was prepared from 12-bromo-l-dodecanol and 3- methylbutylbromide.
  • the 1 H NMR was identical to that reported by Masuda et al.
  • 17-methyloctadecan-l-ol was prepared from 12-bromo-l-dodecanol and 5- methylhexylbromide in 44% yield.
  • branched alkoxyalkanols (6) Preparation of branched alkoxyalkanols (6).
  • branched alkoxyalkanols were prepared by conversion of the branched methyl alkanols (4) to the corresponding methanesulfonate derivatives (5), followed by reaction with either 1,3-propanediol or 1,2-ethanediol.
  • Branched methylalkoxyalkyl esters were prepared from cyclic phosphonates as shown in Scheme 2 using cyclic cidofovir for purposes of illustration. Briefly, the cyclic phosphonates were coupled to branched methylalkoxyalkanols using the Mitsunobu reaction as described by Wan et al. ((2005) Antimicrobial Agents and Chemotherapy 49:656-662) to form the cyclic diesters which were then hydrolyzed to form the branched methylalkoxyalkyl esters.
  • Reagents a) triphenylphosphine, diisopropyl azodicarboxylate, N 5 N-DMF; b) 2 M aq. NaOH, 8O 0 C
  • reaction mixture was heated at 8O 0 C for 2 hrs at which time the TLC showed complete consumption of 11.
  • the reaction was then cooled to room temperature and quenched with dropwise addition of saturated aq. NH 4 Cl. 200 mL of water was added and the target product was extratcted with EtOAc (3x), the combined organic layer was successively washed with water (3x), brine (Ix) and dried over MgSO 4 .
  • the solvent was evaporated under reduced pressure and the residue was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 23 g of 12 as colorless oil in 85% yield.
  • Branched methyl esters were prepared from the p- toluenesulfonyloxymethylphosphonates 18 as illustrated in Scheme 4, using synthesis of (S)-HPMPA esters for purposes of illustration. The procedure is based on the method reported by Beadle et ah, J. Med. Chem. 49:2010-2015, 2006.
  • Reagents a) NaH, N,N-DMF, 50 0 C; b) 80% aq. acetic acid
  • (S)-9-[3-trityloxy-2-hydroxypropyl]-N6-trityl- adenine 17 was prepared from adenine and (S)-trityl glycidyl ether (Daiso Co., Ltd., Japan) following the method of Webb ((1989) Nucleosides & Nucleotides 8:619-624). Sodium hydride (24 mg, 1.0 mmol) was added to a stirred solution of (S)-9-[3-trityloxy- 2-hydroxypropyl]-N 6 -trityladenine (640 mg, 0.62 mmol) in dry triethylamine (10 mL).
  • 2- bromopropanoic acid 35 is reduced to the alcohol with borane:THF complex solution to provide 2-bromo-l-propanol 36.
  • Fluorination of 36 is achieved with l,l,2-trifluoro-2- chloroethyldiethylamine, a mild and safe reagent to convert l-hydroxy-2- halogenoalkanes into the corresponding rearranged fluoride 37.
  • Conversion of 37 into a Grignard reagent followed by reaction with 13-bromo-tridecanol in the presence of the catalyst provides 15-fluorohexadecanol 38.
  • step a Conversion of alcohol 38 into the methanesulfonate derivative, followed by reaction with 1,3-propanediol provides 3-(15- fluorohexadecyloxy)propan-l-ol 39.
  • step b Reaction of 39 with cyclic cidofovir or cyclic (S)- HPMPA as described generally in Example 2 (step a), provides the cyclic esters (40 and 41, respectively) which can then be converted to the desired compounds (42 and 43, respectively) using the general method set forth in Example 2 (step b).
  • Reagents a) BH 3 , THF; b) fluoroamine; c) Mg/13-bromo-tridecanol, Li 2 CuCl 4 ; d) methanesulfonyl chloride, pyridine, then 1,3 -propanediol, NaH, N 5 N-DMF; e) cCDV or cHPMPA, DIAD, PPh 3 , N 5 N-DMF; f) 1 M NaOH
  • the Grignard reagent prepared from l-bromo-4-fluorobutane and magnesium is reacted with 12-bromododecanol 44 to obtain 16-fluorohexadecanol 45.
  • Reaction of 45 with methanesulfonyl chloride followed by reaction with 1,3-propanediol provides 16-fluorohexadecyloxy-l-propanol 46.
  • step a Coupling of 46 with cyclic cidofovir or cyclic (S)-HPMPA as described generally in Example 2 (step a), provides the cyclic esters (47 and 48, respectively) which can then be converted to the desired compounds (49 and 50, respectively) using the general method set forth in Example 2 (step b).
  • Reagents a) Mg/l-bromo-4-fluorobutane, Li 2 CuCl 4 , THF; b) methanesulfonyl chloride, pyridine; c) 1,3-propanediol, NaH, N 5 N-DMF; d) cCDV or cHPMPA, DIAD, PPh 3 , N 5 N-DMF; e) IM NaOH
  • the reaction mixture was stirred for 3 h after which it was diluted with 100 mL of DCM and successively washed with saturated solution of NaHCO 3 , H 2 O and brine, dried over MgSO 4 , filtered and evaporated to dryness.
  • the residue was purified using combiflash (120 g silica column) with hexane/ethyl acetate as solvents to furnish 18 g (90%) of the target product 52 as colorless oil.
  • reaction was then cooled in an ice bath and quenched with a solution of saturated aq. NH 4 Cl. 300 mL of water was then added and reaction mixture was extracted with DCM (4x). The combined DCM layers were then washed with H 2 O (3x), brine (Ix), dried over MgSO 4 , filtered and the solvent was evaporated under vacuum. The residue was then purified using combiflash (120 g silica column) with hexane/ethyl acetate as eluent to furnish 12.86 g (72%) of the target product 54 as colorless oil.
  • Example 10 Evaluation of antiviral activity of penultimate branched methyl alkoxyalkyl analogs of cidofovir (CDV) and 9-(S)-(3-hydroxy-2- phosphonomethoxypropyP-adenine ((S)-HPMPA) against vaccinia virus and cowpox virus in vitro
  • Virus pool preparation The vaccinia virus strain, Copenhagen, and cowpox virus, strain Brighton, stock pools were obtained from John Huggins of the U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Md. These pools were prepared in Vero cells and were diluted 1:50 to provide working stocks.
  • Plaque reduction assay for efficacy Two days prior to use, HFF cells were plated on six-well plates and incubated at 37°C with 10% CO 2 and 90% humidity. On the day of the assay, the drugs were made up at twice the desired concentration in 2x minimal essential medium (MEM) containing 5% fetal bovine serum (FBS) and antibiotics and diluted serially 1:5 in 2x MEM to provide six concentrations of drug.
  • MEM minimal essential medium
  • FBS fetal bovine serum
  • the initial starting concentration was usually 200 ⁇ M and ranged down to 0.06 ⁇ M.
  • the virus to be used was diluted in MEM containing 10% FBS to a desired concentration which would give 20 to 30 plaques per well.
  • the medium was then aspirated from the wells, and 0.2 mL of virus was added to each well in triplicate, with 0.2 mL of medium being added to drug toxicity wells.
  • the plates were incubated for 1 h with shaking every 15 min. After the incubation period, an equal amount of 1% agarose was added to an equal volume of each drug dilution. This gave final drug concentrations beginning with 100 ⁇ M and ending with 0.03 ⁇ M and a final agarose overlay concentration of 0.5%.
  • the medium was aspirated and 125 ⁇ L of drug was added to the first row of wells and then diluted serially 1:5 using the Beckman BioMek liquid-handling system. After the addition of the drug, the plates were incubated for 7 days in a CO 2 incubator at 37°C. At that time, the medium with drug was aspirated, and 200 ⁇ l of 0.01% neutral red in phosphate-buffered saline (PBS)/well was added and incubated for 1 h. The dye was aspirated, and the cells were washed with PBS using a Nunc plate washer. After the PBS was removed, 200 ⁇ L of 50% ethanol-1% glacial acetic acid (in H 2 0)/well was added.
  • PBS phosphate-buffered saline
  • CDV cidofovir
  • HDP-CDV hexadecyloxypropyl-CDV
  • 17M-ODE-CDV 17-methyl- octadecyloxyethyl cidofovir
  • 15M-HDP-CDV 15-methyl-hexadecyloxypropyl cidofovir
  • 14M-PDP- CDV 14-methyl-pentadecyloxypropyl cidofovir
  • 13M-TDP-CDV 13-methyl-tetradecyloxypropyl cidofovir
  • 12M-TrDP-CDV 12-methyl-tridecyloxypropyl cidofovir
  • BS-C-I cells (ATCC CCL 26) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine serum fetal clone III (Hyclone, Logan, UT), 2 mM L-glutamine (GIBCO, Grand Island, NY), 100 U/mL penicillin (GIBCO), and 100 ⁇ g/mL streptomycin (GIBCO).
  • DMEM Dulbecco's modified Eagle's medium
  • a plaque-purified isolate of the MOS strain of ECTV (ATCC VR-1374) designated MOS-3-P2 was propagated in an African green monkey kidney cell line, BS-C-I. Virus was purified through a sucrose cushion.
  • Virus suspensions were serially diluted in PBS + 1% sera, absorbed to monolayers for 1 h at 37°C, and overlayed with a suspension of 1% carboxyl methyl cellulose in DMEM +5% Fetal clone III. After 4 days at 37°C, virus plaques were visualized and virus inactivated by the addition to each well of 0.5 mL of a 0.3% crystal violet/10% formalin solution.
  • Plaque reduction assay Plaque reduction assay. CV-I cells were plated in wells of a 24- well cluster plate. Each monolayer was infected with 75 plaque forming units (PFU) of indicator virus in 0.1 mL of DMEM +5% Fetal clone III for 60 min at 37 0 C. Media was removed by aspiration and standard virus overlay media containing no drug or the test drug at concentrations ranging from 0.05 to 50 ⁇ M was added. The plates were incubated at 37°C for 3-4 days for ECTV and 2 days for VACV-WR, monolayers were stained, and plaques counted using a stereomicroscope. The EC 50 concentration for each drug was calculated. The results are set forth in Table 5.
  • Example 12 Activity of oral 15M-HDP-(S)-HPMPA in lethal ectromelia virus infection
  • mice were injected to effect with an anesthetic cocktail (ketamine 90 mg/kg / xylazine 10 mg/kg), held at 45° C from the vertical on a intubation platform, and inoculated with 5 ⁇ l of virus suspension in each naris for a total challenge dose of 140 PFU (-280 xLD50). Approximately 2.5 min following inoculation of virus, the mice were returned to their cage. Four hours following exposure to ECTV, groups of mice were treated by gavage with 0.1 ml of sterile, distilled water alone or water containing the test compound. This treatment was repeated on days 1, 2, 3, and 4 for a total of five doses. The mice were observed over 21 days for clinical signs of disease (morbidity) and mortality. The results are set forth in Table 6.
  • HDP hexadexyloxypropyl
  • 15M-HDP 15-methyl-hexadecyloxypropyl
  • 13M-TDP 13-methyl-tetradecyloxypropyl
  • 14M-PDP 14-methyl-pentadecyloxypropyl.
  • MDD Mean day of death
  • Target compounds were incubated in pooled S9 liver fractions from monkey and human liver (purchased from a commercial source) at 37 0 C.
  • the control sample (immediately quenched) was used to determine the response at time zero.
  • the ratio between the response of the incubated samples and time zero indicated the % parent compound remaining.
  • Compounds were dissolved in DMSO and serially diluted with buffer to a concentration suitable for the assay (1 to 10 ⁇ M). A portion of a high concentration dilution (-500 mM) was used to determine the LC-MS/MS conditions for analysis (ionization polarity, SRM transition, collision energy). 7-ethoxycoumarin was included as a control.
  • Negative controls (without S9) were included to check the stability of the test compounds at incubation conditions. All assays were performed in triplicate and the % parent compound remaining was reported. Incubations were generally performed in microtiter plates with a protein concentration of 3 mg/mL and a compound concentration of 1 ⁇ M. Reactions were sampled at the specified time points and stopped by the addition of a cold acetonitrile/water solution. The quenched plates were centrifuged and subsequently analyzed by fast gradient LC-MS/MS. The results are set forth in Figure 3.

Abstract

The present invention relates to phosphonate, nucleoside phosphonate or nucleoside phosphate compounds, compositions containing them, processes for obtaining them, and their use in treating a variety of medical disorders, in particular viral infections, cancers and the like.

Description

METABOLICALLY STABLE ALKOXYALKYL ESTERS OF
ANTIVIRAL OR ANTIPROLIFERATIVE PHOSPHONATES,
NUCLEOSIDE PHOSPHONATES AND NUCLEOSIDE
PHOSPHATES
FIELD OF THE INVENTION
[0001] The present invention relates to orally administered drugs for treatment of viral infections and certain cancers. In particular, the present invention relates to metabolically stable alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates, compositions containing them, processes for obtaining them, and their use in treating a variety of medical disorders, in particular viral infections, cancers and the like.
BACKGROUND OF THE INVENTION
[0002] Nucleoside phosphonates have antiviral, antiproliferative and a variety of other therapeutic benefits. Among these are the antiviral nucleoside phosphonates, such as, for example, cidofovir, cyclic cidofovir, adefovir, tenofovir, and the like, as well as the 5'-phosphonates and methylene phosphonates of azidothymidine (AZT), ganciclovir, acyclovir, and the like. In these compounds, the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides (ganciclovir, penciclovir, acyclovir) which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond. In the case of the methylene phosphonates, a methylene group replaces the 5'- hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate.
[0003] Upon cellular metabolism of nucleoside phosphonates, two additional phosphorylations occur to form the nucleoside phosphonate diphosphate which represents the equivalent of nucleoside triphosphates. Antiviral nucleoside phosphonate diphosphates are selective inhibitors of viral RNA or DNA polymerases or reverse transcriptases. That is to say, their inhibitory action on viral polymerases is much greater than their degree of inhibition of mammalian cell DNA polymerases α, β and γ or mammalian RNA polymerases. Conversely, the antiproliferative nucleoside phosphonate diphosphates inhibit cancer cell DNA and RNA polymerases and may show much lower selectivity versus normal cellular DNA and RNA polymerases. [0004] As noted above, one class of antiviral and antiproliferative compounds are the antiviral nucleoside phosphonates. Two representative structures of this class of compounds, namely CDV and HPMPA, are set forth below:
cidofovir HPMPA
[0005] Another class of phosphonates is the 5'-phosphonates and methylene phosphonates of azidothymidine, ganciclovir, acyclovir, and the like. In compounds of this type, the 5'-hydroxyl of the sugar moiety, or its equivalent in acyclic nucleosides (ganciclovir, penciclovir, acyclovir), which do not contain a complete sugar moiety, is replaced with a phosphorus-carbon bond. In the case of the methylene phosphonates, a methylene group replaces the 5'-hydroxyl or its equivalent, and its carbon atom is, in turn, covalently linked to the phosphonate. Two representative structures of this class of compounds, namely AZT 5'-phosphate and AZT 5'-phosphonate, are set forth below.
AZT 5'-methylene AZT 5'-phosphonate phosphonate
[0006] Another class of therapeutically effective compounds is the nucleoside phosphates, such as, acyclovir monophosphate, 2'-O-methyl-guanosine-5'-phosphate, 2 '-O-methyl-cytidine-5 '-phosphate and 2 '-C-methyl-cytidine-5 '-phosphate. Two representative structures of this class of compounds are set forth below:
Acyclovir monophosphate
2'-C-methyl-cytidine-5'-phosphate
[0007] Yet another class is the antiviral phosphonates, phosphonoformate and phosphonoacetate as illustrated below.
1 -O -octadecyl -2-0- methyl- sn-glycero-3-phos- l-O-octadecyl-2-O-methyl-sn-glycero-3-phos- phonoformate, ethyl ester phonoacetate, ethyl ester
[0008] Various substituent groups may be attached to phosphonates and phosphates to produce derivatives having various degrees of pharmacological potency. One class of derivative compounds are the alkoxyalkyl esters, such as hexadecyloxypropyl cidofovir (HDP-CDV), which is illustrated by the following general structure:
Hexadecyloxypropyl cidof ovir (HDP-CDV) .
CDV itself is not orally active; however esterification of CDV with certain alkoxyalkanols such as hexadecyloxypropanol dramatically increases its antiviral activity and selectivity in vitro and confers a degree of oral bioavailability. The alkyl chain length of these CDV analogs is related to solubility and the ability of the compounds to associate with biomembranes.
[0009] Although alkoxyalkyl esters of nucleoside phosphates and phosphonates, such as hexadecyloxypropyl-cidofovir (HDP-CDV), have therapeutically beneficial properties, they suffer from pharmacological disadvantages as orally administered agents. Orally administered drugs are usually taken up from the small intestine into the portal vein, which exposes the drug to potentially rapid lipid metabolism in the enterocytes of the small intestines and in the liver. Alkoxyalkyl esters of phosphates and phosphonates, such as HDP-CDV can be incorporated into cell membranes where the phosphate or phosphonate is subsequently liberated inside the cell or can be oxidatively metabolized by the cytochrome P450s such as CYP3A4 in the liver or intestine leading to omega oxidation of the alkyl chain followed by beta oxidation. It has recently been determined that alkoxyalkyl esters of phosphates and phosphonates can be oxidized at the terminal end of the alkyl chain by omega oxidation and are further degraded by beta oxidation to short chain inactive metabolites. This process, which is illustrated in Figure 1 for nucleoside monophosphonate HDP-CDV, may be very rapid and is deleterious to the intended pharmacologic effect of the compounds. In the case of HDP-CDV, the inactive metabolite is water soluble, virologically inactive, and rapidly excreted in the urine. Rapid metabolism by this pathway may lower plasma levels of the prodrug, and reduce the antiviral efficacy of HDP-CDV and alkoxyalkyl esters of phosphonates, nucleoside phosphonates and nucleoside phosphates. [0010] There is therefore a continuing need for more stable pharmaceutical agents to treat a variety of disorders, such as those caused by viral infection and inappropriate cell proliferation, e.g. cancer. Thus, it is an object of the present invention to develop chemically modified phosphonates, nucleoside phosphonates and nucleoside phosphates that can slow the metabolism of oral antiviral and anticancer compounds.
SUMMARY OF THE INVENTION
[0011] The present invention includes esters of phosphonates, nucleoside phosphonates and nucleoside phosphates (referred to collectively hereinafter as esters) that are resistant to metabolic inactivation resulting from oxidation of these compounds in the liver. More specifically, the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted alkoxyalkyl esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing metabolic stability during absorption in the small intestine, first pass liver metabolism and subsequent distribution to peripheral tissues. Included in the present invention are methods for using said esters for treating various diseases and conditions. [0012] The compounds and methods of this invention are based upon the unique insight that ω-oxidation of lipid esters of phosphonates and phosphates may be slowed by placing a blocking group or groups at or near the penultimate carbon of the alkyl chain. Potential blocking groups include, but are not limited to alkyl groups, including, but not limited to methyl, ethyl and propyl, cyclopropyl and halogens. Potential blocking groups also include alkenyl groups containing one or more double bonds, including a terminal double bond. Although substituted alkoxyalkyl phosphates and alkylglycerol phosphates are known in the art, the use of penultimate or terminally substituted alkyl chains to stabilize lipid phosphate or phosphonate ester drugs against rapid omega and beta oxidation has not been reported previously. Phosphonate compounds contemplated for use in accordance with the present invention include those having antiviral and antiproliferative activity.
[0013] Representative examples of the phosphonate compounds and esters thereof contemplated for use in accordance with the present invention are set forth in the references cited in Table 1. Also included within the scope of the instant invention are nucleoside analogs with antiviral activity against hepatitis C, which can be converted to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates. Examples of nucleosides in this class of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine. Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'-phosphates or their alkylglycerol phosphates are set forth in the references cited in Table 2.
[0014] Further included are nucleoside analogs with antiviral activity against hepatitis B, which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates. Exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various β-L-2'- deoxycytidine, β-L-2'-deoxyadenine and β-L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45[l):229-235). [0015] Anticancer agents may also be derivatized according to the method of this invention. Some subject compounds include but are not limited to (E)-2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- β-D- arabinosyl)cytosine (4'-thio-FAC). Other antiproliferative nucleosides may also become active when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol. Non- nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl groups of the invention including, but not limited to topotecan by phosphorylating and esterifying an available hydroxyl group. Etoposide may be derivatized by attaching metabolically stable groups of the invention to the phosphate residue of etoposide.
[0016] Phosphonate and phosphate analogs contemplated for use in accordance with the present invention are selected to improve the bioactivity, selectivity, and/or bioavailability of the antiviral or antiproliferative compounds.
[0017] In another aspect of the present invention, there are provided pharmaceutical formulations containing the analogs of the phosphonate compounds described herein. [0018] In yet another aspect of the present invention, there are provided a variety of therapeutic methods, e.g. methods for treating viral infections and methods for treating disorders caused by inappropriate cell proliferation, e.g. cancer and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 illustrates schematically antiviral activation and metabolic inactivation pathways for hexadecyloxypropyl cidofovir (HDP-CDV).
[0020] Figure 2 depicts representative structures of "Metabolism Resistant" lipophilic esters of cidofovir.
[0021] Figure 3 depicts a graph of the % of drug remaining versus time for HDP-
CDV and 15-methyl-HDP-CDV (15M-HDP-CDV). This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative
HDP-CDV. The methods are described in Example 13.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0022] The present invention includes chemical methods for synthesizing alkoxyalkyl esters having certain moieties at or near the omega end of the alkyl chain which block or slow degradation and metabolic inactivation. Specifically, the present invention includes terminal or penultimate branched chain, unsaturated and halogen substituted esters of phosphonate compounds, wherein said substituents stabilize these compounds by providing resistance to oxidation. Phosphonates, nucleoside phosphonates and nucleoside phosphates having antiviral or anticancer activity are subjects of the invention.
[0023] Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided.
[0024] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a phosphonate refers to one or more phosphonates. As such, the terms "a" or "an", "one or more" and "at least one" are used interchangeably herein. [0025] It is also to be noted that in some cases for purposes of illustration only a single stereoisomer is depicted for a particular compound. However, the method of the invention is not limited to any particular isomer and can be extended to the S enantiomer, the R enantiomer or racemic mixtures thereof.
[0026] As used herein, the term "prodrug" refers to derivatives of pharmaceutically active compounds that have chemically or metabolically cleavable groups and become the pharmaceutically active compound by solvolysis or under in vivo physiological conditions.
[0027] The term "purine or pyrimidine base" includes, but is not limited to, 6- alkylpurine and N6-alkylpurines, N6-acylpurines, N6-benzylpurine, 6-halopurine, N6- acetylenic purine, N -acyl purine, N -hydroxyalkyl purine, 6-thioalkyl purine, N - alkylpurines, 7-deazapurines, N4-alkylpyrimidines, N4-acylpyrimidines, A- halopyrimidines, N4-acetylenic pyrimidines, 4-amino and N4-acyl pyrimidines, A- hydroxyalkyl pyrimidines, 4-thioalkyl pyrimidines, thymine, cytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil, C -alkylpyrimidines, C5-benzylpyrimidines, C5-halopyrimidines, C5-vinylpyrimidine, C5-acetylenic pyrimidine, C5-acyl pyrimidine, C5-hydroxyalkyl purine, C5-amidopyrimidine, C5- cyanopyrimidine, C5-nitropyrimidine, C5-aminopyrimidine, N2-alkylpurines, N2-alkyl- 6-thiopurines, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl. Included in this definition are ring- expanded and open-ring cogeners of any of the aforementioned purines. Functional oxygen and nitrogen groups on the base can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p- toluenesulfonyl. Preferred bases include cytosine, 5-fluorocytosine, uracil, thymine, adenine, guanine, xanthine, 2,6-diaminopurine, 6-aminopurine, 6-chloropurine and 2,6- dichloropurine.
[0028] The term "alkyl" as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. The alkyl group can be optionally substituted with one or more halogens selected from the group consisting of F, Cl, Br or I.
[0029] The term "alkenyl" as used herein, unless otherwise specified, refers to a partially unsaturated straight or branched hydrocarbon. The alkenyl group can be optionally substituted with one or more halogens selected from the group consisting of
F, Cl, Br or I.
[0030] The term "protected" as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis. Suitable protecting groups are described, for example, in Greene, et al., "Protective Groups in Organic Synthesis,"
John Wiley and Sons, Second Edition, 1991, which is incorporated herein by reference in its entirety.
[0031] The nucleoside phosphonates of the instant invention can be generally represented by the following structures.
wherein
R is selected from the group consisting of -R1-O-R2, wherein R1 is selected from the group consisting of an optionally substituted C1 to C11 alkyl group and R2 is selected from the group consisting of a C6 to Cn alkyl group or a C6 to Cn alkenyl group; wherein said Ce to Cn alkyl group is substituted with one or more alkyl groups selected from the group including, but not limited to methyl, ethyl, propyl, or cycloalkyl, including, but not limited to cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said C6 to C17 alkyl group includes one or more substituents at or near the terminal position of the alkyl group, in particular at the terminal or penultimate position; and wherein said Ce to Cn alkenyl group is optionally-substituted with an alkyl group selected from the group including, but not limited to methyl, ethyl, propyl, a cycloalkyl group including, but not limited to cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein the said C6 to Cn alkenyl group contains one or more double bonds, including a terminal double bond;
B is selected from a purine or pyrimidine base; and
A is a counterion selected from the group including, but not limited to H+, Li+, Na+, K+,
NH4 +, tetraalkyl ammonium and other tertiary amine salts including but not limited to triethylamine.
[0032] In one embodiment R is selected from the group of compounds having the general structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17.
[0033] In another embodiment R is selected from the group of compounds having the general structure:
wherein p and q are as defined above.
[0034] In yet another embodiment R is selected from the group of compounds having the general structure:
wherein p and q are as defined above and X is a halogen. In preferred embodiments X is F.
wherein p and q are as defined above and X is independently selected from a halogen. In preferred embodiments X is F. [0035] In specific embodiments R is selected from the group consisting of one of the structures set forth in Figure 2.
[0036] In one embodiment of the invention derivatized nucleoside phosphonates are analogs of cyclic cidofovir or cidofovir which can be generally represented by the following structures:
cyclic cidofovir cidofovir wherein R and A are as defined above.
[0037] In another embodiment of the invention the derivatized nucleoside phosphonates are analogs of 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)-adenine ((S)-HMPMA) which can be generally represented by the following structure:
cyclic (S)-HPMPA (S)-HPMPA wherein R and A are as defined above.
[0038] Specific analogs of cyclic cidofovir, cidofovir and HPMPA included in the present invention include the following compounds: 3-(12-methyltridecyloxy)propyl cyclic cidofovir, 3-(13-methyltetradecyloxy)propyl cyclic cidofovir, 3-(14- methylpentadecyloxy)propyl cyclic cidofovir, 2-(17-methyloctadecyloxy)ethyl cyclic cidofovir, 3-(15-methylhexadecyloxy)propyl cyclic cidofovir, 3-(15- methylhexadecyloxy)ethyl (S)-cyclic HPMPA, 3-(15-methylhexadecyloxy)propyl (S)- cyclic HPMPA, 2-(17-methyloctadecyloxy)ethyl-(S)-cyclic HPMPA, 3-(12-methyl- tridecyloxy)propyl cidofovir, 3-(13-methyl-tetradecyloxy)propyl cidofovir, 3-(14- methyl-pentadecyloxy)propyl cidofovir, 3-(15-methyl-hexadecyloxy)propyl cidofovir, sodium, 3-(15-methyl-hexadecyloxy)propyl cidofovir, ammonium, 2-(17-methyl- octadecyloxy)ethyl cidofovir, 2-(15-methyl-hexadecyloxy)ethyl cidofovir, 3- (phytanyloxy)propyl cidofovir, 3-(15-methylhexadceyloxy)ethyl-(S)-HPMPA and 2- (17-methyloctadecyloxy)ethyl-(S)-HPMPA, 3-(hex-dec-15-enyloxy)propyl cidofovir, ammonium, 3-(15-fluorohexadecyloxy)propyl cidofovir, 3-(15- fluorohexadecyloxy)propyl cyclic cidofovir, 3-(15-fluorohexadceyloxy)propyl-(S)- HPMPA, 3-(15-fluorohexadceyloxy)propyl-(S)-cyclic HPMPA, 3-(16- fluorohexadecyloxy)propyl cidofovir, 3-(16-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(16-fluorohexadceyloxy)propyl-(S)-HPMPA, 3-(16- fluorohexadceyloxy)propyl-(S)-cyclic HPMPA and l l-(7, 7, 8, 8, 8-pentafluoro- octyloxy)undecyl-cidofovir, ammonium.
[0039] The nucleoside phosphates and analogs thereof of the instant invention can be generally represented by the following structures.
wherein R is an alkoxyalkyl group having a structure as defined above and B is a substituted or unsubstituted pyrimidine base or their open ring congeners. [0040] Representative examples of nucleosides in this group of compounds include, but are not limited to 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7-deaza-2'-methyl adenosine, 2'-C-methyl cytosine. Other nucleosides and analogs thereof contemplated for use in accordance with this invention following conversion to their alkoxyalkyl 5'- phosphates or their alkoxyalkylglycerol phosphates are set forth in the references cited in Table 2.
[0041] Further included are nucleoside analogs with antiviral activity against hepatitis B, which may be converted to their 5'-phosphates, 5'-phosphonates or 5'-methylene phosphonates. Exemplary nucleosides in this class of compounds include, but are not limited to 3TC, FTC, DAPD, L-FMAU, entecavir, telbivudine and various β-L-2'- deoxycytidine, β-L-2'-deoxyadenine and β-L-2'-deoxythymidine analogs described by Bryant et al. ((Jan. 2001) Antimicrob Agents Chemother 45£l):229-235). Phosphates of non-nucleoside antivirals are also subjects of the invention including, but not limited to, zanamivir (Relenza®).
[0042] Anticancer agents may also be derivatized according to the method of this invention. Some representative compounds include, but are not limited to 2'-deoxy-2'- fluoromethylene-cytidine (FMdC) and l-(2-deoxy-2-fluoro-4-thio- β-D- arabinosyl)cytosine (4'-thio-FAC). Other antiproliferative nucleosides may also become more metabolically stable when derivatized according to the invention including, but not limited to Ara-C, Ara-G, 5-fluorouridine, 5-fluoro-deoxyuridine, fludarabine, gemcitabine, decitabine or alkylglycerol phosphate or alkoxyalkyl phosphate esters of taxol. Non-nucleoside cancer agents may be similarly derivatized with the metabolically stable alkoxyalkyl esters of the invention including, but not limited to topotecan by coupling to an available hydroxyl group. Etoposide may be coupled to the metabolically stable alkoxyalkyl esters of the invention by attachment to the phosphate residue of etoposide.
[0043] Tables 1 and 2 provide examples of compounds, which may be subjected to the chemical steps of the invention. The references cited in these Tables are hereby incorporated by reference in their entirety. Table 1. References citing illustrative phosphonates, nucleoside phosphonates and nucleoside phosphates for use according to the method of this invention
Table 2. References citing illustrative analogs of nucleosides, which can be converted to nucleoside hos hates for use accordin to the method of this invention
[0044] Compounds of the instant invention can be administered orally in the form of tablets, capsules, solutions, emulsions or suspensions, inhaled liquid or solid particles, microencapsulated particles, as a spray, through the skin by an appliance such as a transdermal patch, or rectally, for example, in the form of suppositories. The lipophilic prodrug derivatives of the invention are particularly well suited for transdermal absorption administration and delivery systems and may also be used in toothpaste. Administration can also take place parenterally in the form of injectable solutions. [0045] The compositions may be prepared in conventional forms, for example, capsules, tablets, aerosols, solutions, suspensions, or together with carriers for topical applications. Pharmaceutical formulations containing compounds of this invention can be prepared by conventional techniques, e.g., as described in Remington's Pharmaceutical Sciences, 1985.
[0046] The pharmaceutical carrier or diluent employed may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene or water. The carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or distearate, alone or mixed with a wax.
[0047] If a solid carrier is used for oral administration, the preparation may be tabletted or placed in a hard gelatin capsule in powder or pellet form. The amount of solid carrier will vary widely, but will usually be from about 25 mg to about I g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution. [0048] Tablets are prepared by mixing the active ingredient (that is, one or more compounds of the invention), with pharmaceutically inert, inorganic or organic carrier, diluents, and/or excipients. Examples of such excipients which can be used for tablets are lactose, maize, starch or derivatives thereof, talc, stearic acid or salts thereof. Examples of suitable excipients for gelatin capsules are vegetable oils, waxes, fats, semisolid, and liquid polyols.
[0049] For nasal administration, the preparation may contain a compound of the invention dissolved or suspended in a liquid carrier, in particular, an aqueous carrier, for aerosol application. The carrier may contain solubilizing agents such as propylene glycol, surfactants, absorption enhancers such as lecithin or cyclodextrin, or preservatives.
[0050] Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous liquids, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
[0051] Suitable excipients for the preparation of solutions and syrups are water, polyols, sucrose, invert sugar, glucose, and the like. Suitable excipients for the preparation of injectable solutions are water, alcohols, polyols, glycerol, vegetable oils, and the like.
[0052] The pharmaceutical products can additionally contain any of a variety of added components, such as, for example, preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, antioxidants, diluents, and the like.
[0053] Optionally, the pharmaceutical compositions of the invention may comprise a compound according to the general formula combined with one or more compounds exhibiting a different activity, for example, an antibiotic or other pharmacologically active material. Such combinations are within the scope of the invention. [0054] This invention provides methods of treating disorders related to viral infections, inappropriate cell proliferation, and the like. The methods particularly comprise administering to a host in need thereof a therapeutically effective amount of the prodrugs of this invention. Thus, in one aspect of the invention there are provided methods for treating disorders caused by viral infections. Indications appropriate to such treatment include susceptible viruses including, but are not limited to human immunodeficiency virus (HIV), influenza, herpes simplex virus (HSV), human herpes virus 6 and 8, cytomegalovirus (CMV), hepatitis B and C virus, Epstein-Barr virus (EBV), varicella zoster virus, and diseases caused by orthopox viruses (e.g., variola major and minor, vaccinia, smallpox, cowpox, camelpox, monkeypox, and the like), ebola virus, papilloma virus, and the like, lymphomas, hematological disorders such as leukemia, and the like, and cancers caused by viruses such as cervical cancer which is caused, in most cases, by the high risk subtypes of human papilloma virus. [0055] In yet another aspect of the invention, there are provided methods for treating disorders caused by inappropriate cell proliferation, e.g. cancers, such as melanoma, lung cancers, pancreatic cancer, stomach, colon and rectal cancers, prostate and breast cancer, the leukemias and lymphomas, and the like. Anti-cancer compounds which can be converted to their nucleotide phosphonates or nucleoside-5 '-phosphates for use as compounds of this invention include, but are not limited to, cytarabine (ara-C), fluorouridine, fluorodeoxyuridine (floxuridine), gemcitibine, decitabine, cladribine, fludarabine, pentostatin (2'-deoxycoformycin), 6-mercaptopurine and 6-thioguanine and substituted or unsubstituted ara-adenosine (ara-A), ara-guanosine (ara-G), and ara- uridine (ara-U). Anticancer compounds of the invention may be used alone or in combination with other antimetabolites or with other classes of anticancer drugs such as alkaloids, topoisomerase inhibitors, alkylating agents, antitumor antibiotics, and the like.
[0056] The prodrugs of the invention can be administered orally, parenterally, topically, rectally, and through other routes, with appropriate dosage units, as desired. [0057] As used herein, the term "parenteral" refers to subcutaneous, intravenous, intra-arterial, intramuscular or intravitaeal injection, or infusion techniques. [0058] The term "topically" encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and mucous membranes of the mouth and nose and in toothpaste.
[0059] "Therapeutic" as used herein, includes treatment and/or prophylaxis. When used, therapeutic refers to humans as well as other animals.
[0060] "Pharmaceutically or therapeutically effective dose or amount" refers to a dosage level sufficient to induce a desired biological result. That result may be the alleviation of the signs, symptoms or causes of a disease or any other alteration of a biological system that is desired.
[0061] A "host" or "patient" is a living subject, human or animal, into which the compositions described herein are administered.
[0062] With respect to disorders associated with viral infections or inappropriate cell proliferation, e.g., cancer, the "effective amount" is determined with reference to the recommended dosages of the antiviral or anticancer parent compound. The selected dosage will vary depending on the activity of the selected compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound(s) at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration, for example, two to four doses per day. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors, including the body weight, general health, diet, time, and route of administration and combination with other drugs, and the severity of the disease being treated.
[0063] Generally, the compounds of the present invention are dispensed in unit dosage form comprising 1% to 100% of active ingredient. The range of therapeutic dosage is from about 0.01 to about 1,000 mg/kg/day with from about 0.10 mg/kg/day to 100 mg/kg/day being preferred, when administered to patients, e.g., humans, as a drug. Actual dosage levels of active ingredients in the pharmaceutical compositions of this invention may be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular patient. [0064] Compounds of the invention can be prepared in a variety of ways, as generally depicted in Schemes 3 to 7 in Examples 1-9. The general phosphonate esterification methods described below are provided for illustrative purposes only and are not to be construed as limiting this invention in any manner. Indeed, several methods have been developed for direct condensation of phosphonic acids with alcohols (see, for example, R. C. Larock, Comprehensive Organic Transformations, VCH, New York, 1989, p. 966 and references cited therein). Isolation and purification of the compounds and intermediates described in the examples can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, flash column chromatography, thin-layer chromatography, distillation or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures are in the examples below. Other equivalent separation and isolation procedures can of course, also be used.
[0065] Example 1 (Scheme 1) outlines a general method for the synthesis of branched alkoxyalkanols having the general formula:
wherein p and q are as defined above.
[0066] Example 2 (Scheme 2) outlines a general method for the synthesis of branched methylalkoxyalkyl esters from cyclic phosphonates. Cyclic cidofovir was used in this Example for purposes of illustration, however this method can be extended to the use of virtually any cyclic phosphonate of interest. The following compounds were prepared using the general methods set forth in Examples 1 and 2: 3-(12-methyl- tridecyloxy)propyl cidofovir, 3-(13-methyl-tetradecyloxy)propyl cidofovir, 3-(14- methyl-pentadecyloxy)propyl cidofovir, 3-(15-methyl-hexadecyloxy)propyl cidofovir, sodium, 2-(17-methyl-octadecyloxy)ethyl cidofovir, 2-(15-methyl-hexadecyloxy)ethyl cidofovir, 3-(15-methylhexadceyloxy)ethyl-(S)-HPMPA, and 2-(17- methyloctadecyloxy)ethyl-(S)-HPMPA.
[0067] Examples 3 and 4 describe the synthesis of two specific branched alkoxyalkyl esters, namely 3-(phytanyloxy)propyl cidofovir and 15-methylhexadecyloxypropyl cidofovir (15-Me HDP-CDV), ammonium, using slight variations of the methods described in Examples 1 and 2.
[0068] Example 5 (Scheme 4) describes a general method for the synthesis of the branched methylalkoxyalkyl esters of the instant invention from p- toluenesulfonyloxymethyl phosphonates. The synthesis of the branched methylalkoxyalkyl ester 3-(15-methyl-hexadecyloxy)propyl (S)-9-[3-trityloxy-2- (phosphonomethoxy)propyl]-N6-trityl-adenine, was described for purposes of illustration. [0069] Example 6 (Scheme 5) outlines a general method for the synthesis of alkenyloxyalkyl esters having a terminal double bond. The nucleoside phosphonate cidofovir was used for purposes of illustration resulting in the synthesis of compound 26, hexadec-15-enyl-oxypropyl-cidofovir.
[0070] Examples 7-9 (Schemes 6-8) outline general methods for the synthesis of various halogenated alkoxyalkyl esters using CDV and HPMPA for purposes of illustration. Synthesis of the following compounds are exemplified: 3-(15- fluorohexadecyloxy)propyl cidofovir, 3-(15-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(15-fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(15- fluorohexadecyloxy)propyl-(S)-cyclic HPMPA, 3-(16-fluorohexadecyloxy)propyl cidofovir, 3-(16-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(16- fluorohexadecyloxy)propyl-(S)-HPMPA, 3-(16-fluorohexadecyloxy)propyl-(S)-cyclic HPMPA and l l-(7,7,8,8,8-pentafluoro-octyloxy)undecyl cidofovir. [0071] Examples 10-12 illustrate the antiviral activity representative penultimate branched methyl alkoxyalkyl esters of CDV and HPMPA. The results are set forth in Tables 3-6. As can be seen in Tables 4 and 5 penultimate branched chain analogs of (S)-HPMPA were highly active against vaccinia and cowpox in vitro and penultimate branched chain alkoxyalkyl cidofovir esters were effective against ectromelia virus in vitro at submicromolar EC50s. As can be seen in Table 6, the branch chain analogs of (S)-HPMPA and CDV were all fully protective against death from lethal poxvirus infection at doses of 5 mg/kg/day or greater. 15M-HDP-(S)-HPMPA appeared to be more active than HDP-(S)-HPMPA.
[0072] The stability of the compounds of the invention is illustrated in Example 13, the data for which is set forth in Figure 3. Figure 3 depicts a graph of the % of drug remaining versus time for HDP-CDV and 15-methyl-HDP-CDV (15M-HDP-CDV) after incubation for various times as indicated in the presence of liver S 9 preparations. This figure illustrates that the degradation of branched alkoxyalkyl ester derivative 15M-HDP-CDV by monkey liver and human liver S 9 fractions is markedly slower than that of the straight chain alkoxyalkyl ester derivative HDP-CDV. Thus, changes in the alkyl chain to mimic the features of phytanic acid (3,7,11,15-tetramethyl-hexadecanoic acid) slow ω oxidation of the 16th carbon. Although, the phytantyl derivative illustrated in Example 13 contains 4 methyl groups it is also sufficient to introduce alkyl substituents or halogens at the penultimate carbon as in the hexadecyl moiety of HDP- CDV (15-methyl-hexadexyloxypropyl-cidofovir, 15M-HDP-CDV). Alternatively, terminal fluorines, cyclopropyl, alkene groups also suffice to increase metabolic stability as described in detail above.
EXAMPLES
[0073] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.
[0074] General Procedures. 1H NMR spectra were recorded on a Varian HG spectrophotometer operating at 300 MHz and are reported in units of ppm relative to internal tetramethylsilane at 0.00 ppm. Analtech silica gel-GF (250 micron) plates were used for thin layer chromatography (TLC). The products were visualized with UV light, phospray (Supelco; Bellefonte, PA, USA) and charring. Flash chromatography was performed with silica gel (E. Merck silica gel 60, 230-400 mesh) or with a CombiFlash system (Teledyne Isco, Lincoln, NB). Mass spectra showing the presence of a molecular ion were obtained using electrospray ionization (MS-ESI) in both positive and negative modes.
Example 1. Preparation of branched alkoxyalkanols
[0075] A general method for the synthesis of branched alkoxyalkanols (6) is illustrated in Scheme 1.
Reagents: a) magnesium, THF; b) Li2CuCl4, THF; c) methanesulfonyl chloride, triethylamine, CH2Cl2; d) 1,2-ethanediol or 1,3-propanediol, NaH, N5N-DMF
Scheme 1
[0076] Preparation of branched methylalkanols (4). With reference to Scheme 1, branched methylalkanols were synthesized from bromoalkanols (3) and branched methyl bromoalkanes (1). The chain elongation procedures are described by Fouquet et al (Fouquet and Sclosser (1974) Angew. Chem. Int. Ed. Engl. 13:82-83). [0077] General procedure. A dry THF solution of alkylmagnesium bromide (2) was prepared from branched methyl alkyl bromide (1, 94.2 mmol) and magnesium (113 mmol) in dry THF (90 mL). To a stirred and cooled solution of bromoalkanol (3, 17.3 mmol) in dry THF (50 mL) was added the resulting Grignard reagent, followed by a solution of Li2CuCl4 (0.12 M in dry THF, 8.0 mL, 0.96 mmol) at -780C under N2 atmosphere. The resulting mixture was allowed to warm to room temperature while stirring overnight. After the reaction mixture had been quenched with saturated aq. NH4Cl, it was extracted with ethyl acetate. The extract was successively washed with water, saturated NaHCO3 and brine, dried with MgSO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (10% ethyl acetate/hexanes) to provide the branched methylalkanols (4). [0078] The following compounds were prepared using this general procedure.
12-methyltridecan-l-ol was prepared from 9-bromononan-l-ol and 3- methylbutylbromide in 51% yield. The 1H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal 19:199-204).
13-methyltetradecan-l-ol was prepared from 10-bromodecanol and 3- methylbutylbromide in 62% yield. The 1H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
19:199-204).
14-methylpentadecan-l-ol was prepared from 9-bromononanol and 5- methylhexylbromide in 55% yield. The 1H NMR and MS-ESI data matched those reported by Yuasa et al. (Yuasa and Tsuruta (2004) Flavour and Fragrance Journal
19:199-204).
15-methylhexadecan-l-ol was prepared from 12-bromo-l-dodecanol and 3- methylbutylbromide. The 1H NMR was identical to that reported by Masuda et al.
((2002) Biosci. Biotech. Biochem. 66:1531-1537).
17-methyloctadecan-l-ol was prepared from 12-bromo-l-dodecanol and 5- methylhexylbromide in 44% yield. 1U NMR δ 0.86 (6 H), 1.10-1.40 (32 H), 3.64
(2H).
[0079] Preparation of branched alkoxyalkanols (6). With reference to Scheme 1, branched alkoxyalkanols were prepared by conversion of the branched methyl alkanols (4) to the corresponding methanesulfonate derivatives (5), followed by reaction with either 1,3-propanediol or 1,2-ethanediol.
[0080] General procedure for preparation of methanesulfonates. To a solution of alkanol 4 (100 mmol) and triethylamine (15.2 g, 150 mmol) in CH2Cl2 (100 mL) was added methanesulfonyl chloride (15 g, 130 mmol) at O0C. The reaction mixture was stirred overnight then poured into ice water and extracted with diethyl ether. The extract was washed with saturated aq. NaHCO3 and brine, dried with MgSO4 and concentrated under reduced pressure to give the branched methyl alkylmethanesulfonate 5 in 79-89% yield. The compound was employed in the next step without further purification.
[0081] General procedure for preparation of alkoxyalkanols. 1,3-propanediol or 1,2- ethanediol (10 mmol) was added carefully to a suspension of sodium hydride (2 mmol) in dry N5N-DMF and stirred for 30 min. To the mixture was then added the branched methyl alkylmethanesulfonate (5, 1 mmol) in dry THF. The mixture was heated to 60 0C for 4 h, and then cooled to room temperature. After the mixture had been added to ice water it was extracted with ethyl acetate, washed with brine and concentrated under reduced pressure. The residue was purified by flash chromatography (20% ethyl acetate/hexanes) to give the branched methylalkoxyalkanols (6). [0082] The following compounds were prepared using these procedures.
3-(12-methyltridecyloxy)propan-l-ol,
3-( 13-methyltetradecyloxy)propan- 1 -ol,
3-(14-methylpentadecyloxy)propan-l-ol,
3-(15-methylhexadecyloxy)propan-l-ol 1H NMR δ 0.86 (d, 6H), 1.15 (m, IH), 1.25 (br s, 26 H), 1.60-1.46 (m, 3H), 1.83 (qt, 2H), 3.43 (t, 2H), 3.61 (t, 2H), 3.78 (t, 2H). MS-ESI (m/z) 315.33 (MH)+
2-(15-methylhexadecyloxy)ethan-l-ol
2-(17-methyloctadecyloxy)ethan-l-ol
Example 2. Preparation of branched methylalkoxyalkyl esters from cyclic phosphonates
[0083] Branched methylalkoxyalkyl esters were prepared from cyclic phosphonates as shown in Scheme 2 using cyclic cidofovir for purposes of illustration. Briefly, the cyclic phosphonates were coupled to branched methylalkoxyalkanols using the Mitsunobu reaction as described by Wan et al. ((2005) Antimicrobial Agents and Chemotherapy 49:656-662) to form the cyclic diesters which were then hydrolyzed to form the branched methylalkoxyalkyl esters.
cyclic cidof ovir
Reagents: a) triphenylphosphine, diisopropyl azodicarboxylate, N5N-DMF; b) 2 M aq. NaOH, 8O0C
Scheme 2
[0084] General procedure for preparation of cyclic diesters (illustrated by compound 7). Anhydrous cyclic cidofovir or cyclic (S)-HPMPA (10 mmol), an alkoxyalkanol (6) (20 mmol) and triphenylphosphine (20 mmol) were dissolved or suspended in anhydrous N,N-dimethylformamide (15 mL) and stirred vigorously under a nitrogen atmosphere. Diisopropyl azodicarboxylate (20 mmol) was added in three portions over 15 min and then the mixture was stirred overnight at room temperature. The solvent was then evaporated under vacuum, and the residue was purified by flash column chromatography (15% EtOHZCH2Cl2). The products were finally recrystallized from p- dioxane. The coupled products were equimolar mixtures of the axial and equatorial diastereomers. [0085] The following compounds were prepared:
3-(12-methyltridecyloxy)propyl cyclic cidofovir
3-(13-methyltetradecyloxy)propyl cyclic cidofovir
3-(14-methylpentadecyloxy)propyl cyclic cidofovir 2-(17-methyloctadecyloxy)ethyl cyclic cidofovir
3-(15-methylhexadecyloxy)propyl cyclic cidofovir MS-ESI (m/z) 558.54 (MH)+
3-(15-methylhexadecyloxy)ethyl (S)-cyclic HPMPA
3-(15-methylhexadecyloxy)propyl (S)-cyclic HPMPA MS-ESI (m/z) 582.37 (MH)+
2-(17-methyloctadecyloxy)ethyl-(S)-cyclic HPMPA MS-ESI (m/z) 596.32 (MH)+
[0086] General procedure for preparation of branched methylalkoxyalkyl esters (illustrated by compound 8). The branched methyl alkoxyalkyl esters of cyclic cidofovir (7) or cyclic (S)-HPMPA were suspended in 2 M NaOH (25 mL /mmol), heated to 8O0C and stirred for Ih, during which time the mixtures became clear. After hydrolysis, the solutions were cooled to 250C and acidified with glacial acetic acid to approximately pH 5. The resulting precipitates were collected by vacuum filtration and dried under reduced pressure. The crude products were purified either by flash column chromatography (20% MeOHZCH2Cl2) or recrystallized from ethanol. [0087] The following compounds were prepared:
3-( 12-methyltridecyloxy)propyl cidofovir
3-( 13-methyltetradecyloxy)propyl cidofovir
3-( 14-methylpentadecyloxy)propyl cidofovir
3-(15-methylhexadecyloxy)propyl cidofovir, sodium MS-ESI (m/z) 598.36 (M + Na)+
2-( 17-methyloctadecyloxy)ethyl cidofovir
2-( 15-methyl-hexadecyloxy)ethyl cidofovir
3-(15-methylhexadceyloxy)ethyl (S)-HPMPA
2-(17-methyloctadecyloxy)ethyl (S)-HPMPA MS-ESI (m/z) 614.30 (MH)+.
Example 3. Preparation of 3-(phvtanyloxy)propyl cidofovir
[0088] 3-(Phytanyloxy)propyl cidofovir was prepared using slight modifications of the general methods set forth in Examples 1 and 2 as specifically set forth below. [0089] Preparation of phytanol. Phytol (2.0 g, 6.7 mmol) was dissolved in ethanol, rhodium 5% on alumina was added and mixture was placed under H2 60 psi and shaken overnight. The reaction mixture was filtered and evaporated to give the desired compound as an oil (2.0 g, 100% yield).
[0090] Preparation of phytanylmethanesulfonate. Phytanol (2.0 g, 6.7 mmol) was dissolved in pyridine and cooled to O0C. Methane sulfonyl chloride (1.15 g, 10 mmol) was added and the reaction mixture was stirred for 4 h, after which the mixture was added to ice water and extracted with ether. The ether layer was evaporated to provide a light brown oil (1.5 g) that was used in the next step without further purification. [0091] Preparation of 3-(phytanyloxy)propan-l-ol. Sodium hydride was added carefully to a solution of 1,3-propanediol (7.6 g, 100 mmol) in anhydrous N5N-DMF (30 mL). Phytanylmethanesulfonate (1.5 g, 4 mmol) was added and the mixture was heated to 6O0C and stirred for 4 h. The reaction mixture was then poured into ice/HiO, extracted with dichloromethane, dried over MgSO4 and evaporated. The residue was purified by flash column chromatography using 20% ethyl acetate/hexanes to give 3- (phytanyloxy)propan-l-ol (1.25 g, 87% yield)
[0092] Preparation of 3-(phytanyloxy)propyl cyclic cidofovir. To a stirred mixture of triphenylphosphine, (524 mg, 2 mmol) cyclic cidofovir (anhydrous, 800 mg, 3 mmol) and phytanyloxypropanol (500 mg, 1.4 mmol) was added diisopropyl azodicarboxylate (404 mg, 2 mmol). The mixture was then stirred overnight at room temperature. Solids were removed by filtration and then the filtrate was concentrated and the residue purified by flash column chromatography. Elution with 10-15% EtOH/dichloromethane afforded the cyclic ester (660 mg, 78%) [0093] Preparation of 3-(phvtanyloxy)propyl cidofovir. Phytanyloxypropyl cyclic cidofovir was hydrolyzed as described in Example 2 to provide the target compound.
Example 4. Preparation of 15-methylhexadecyloxypropyl cidofovir (15-Me HDP- CDV)
[0094] 15-Me HDP-CDV, ammonium (16) was prepared using slight modifications of the general methods set forth in Examples 1 and 2 as specifically set forth below and outlined in Scheme 3. TsO(CH2) 14CH(CH3)2 U
— 0TBDMS, TBDMSO" ^ "Q(CH9) 14CH(CH,)9 THF/TBAF
NaH, 900C, 70%
12 lhr 85%
33_N^ MsQ ^\ ^^^^ O(CH2)14CH(CH3)2 14
NH4 + 16
Scheme 3
[0095] 15-Methylhexadecan-l-ol (10). Into a clean flame dried and Ar flushed 500 rnL RBF was added 5 g (33 mmol) of commercially available 9. To this was added 40 mL of anhydrous THF and 970 mg (40 mmol) of Mg turnings, a pellet of I2 was added to accelerate the reaction. The reaction mixture was refluxed for 2 hrs. It was then cooled to room temperature and further to -780C. To this was added 1.6g (6.0 mmol) of 12-bromo-l-dodecanol in 10 mL of anhydrous THF followed by addition of 3.3 mL (0.33 mmol) of lithium cuprate. The resulting mixture was allowed to warm to room temperature while stirring overnight followed by quenching with saturated aq. NH4Cl and extracted with EtOAc (3x). The combined EtOAc layer was then successively washed with water, saturated aq. NaHCO3 and brine, dried over MgSO4 and the solvents evaporated under reduced pressure. The residue was then purified by combiflash using hexane/EtOAc as eluent to furnish 1.07 g of the compound 10 as a white solid in 65-70% yield.
[0096] 15-Methylhexadecyl-l-tosylate (ll). 22 g (86 mmol) of 10 was dissolved in 200 rnL of dry DCM and 14 rnL (100 mmol) of Et3N was added. The solution was cooled to O0C followed by addition of 19 g (100 mmol) of TsCl. The reaction mixture was then stirred for 6 hrs at room temperature and washed with saturated solution of aq. NaHCO3, the organic layer was then dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was then purified by combiflash using hexane/EtOAc as eluent and recrystallized over hexanes at -2O0C overnight to provide 25 g of 11 as white crystals in 70% yield.
[0097] tert-Butyldimethyl-3-(15-methylhexadecyloxy)propoxysilane (12). Into a clean flame dried and Ar flushed 2L RBF was added 22.5 ml (105 mmol) of commercially available 3-tert-butyldimethylsilyloxy propanol and 200 mL of anhydrous DMF was added. The mixture was cooled to O0C and 5.0 g of NaH was added slowly. After complete addition the reaction mixture was stirred at room temperature for 30 minutes and cooled to O0C. 27g (65.7 mmol) of 11 was then slowly added to the reaction with vigorous stirring. After complete addition the reaction mixture was heated at 8O0C for 2 hrs at which time the TLC showed complete consumption of 11. The reaction was then cooled to room temperature and quenched with dropwise addition of saturated aq. NH4Cl. 200 mL of water was added and the target product was extratcted with EtOAc (3x), the combined organic layer was successively washed with water (3x), brine (Ix) and dried over MgSO4. The solvent was evaporated under reduced pressure and the residue was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 23 g of 12 as colorless oil in 85% yield. [0098] 3-(15-methylhexadecyloxypropan-l-ol (13). To 23g (54 mmol) of 12 was added 216 mL of a 1 M solution of TBAF in THF and the reaction was stirred for 16 hrs at room temperature. The reaction mixture was then quenched with saturated solution of aq. NH4Cl and the THF was evaporated under reduced pressure. The aq. solution was then diluted with 200 mL of water and the target product was extracted with Et2O (3x), the combined organic layer was then washed with brine (Ix), dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was then purified by combiflash using hexane/ethyl acetate as eluent to furnish 11.5 g of compound 13 in 70% yield as yellow oil.
[0099] Methanesulfonic acid-3-(15-methylhexadecyloxy)propyl ester (14). To 11 g (35 mmol) of 13 was added 25 rnL of DCM and 7.3 rnL (53 mmol) of Et3N and the mixture was cooled to O0C. MsCl 3.0 mL (38.5 mmol) and a catalytic amount of DMAP was then added dropwise and the reaction was stirred at room temperature overnight. The reaction mixture was then diluted with 50 mL of DCM and washed successively with saturated solution of aq. NaHCO3 (Ix), water (Ix) and brine (Ix), dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was then purified by combiflash using hexane/ethyl acetate as eluent to furnish 13 g of product 14 in quantitative yield.
[00100] Reaction of 14 with Bz-c-CDV. 2.4 g (6.6 mmol) of Bz-c-CDV was dissolved in 10 mL of NMP and 1.7 mL (lOmmol) of DIPEA was added to it followed by 13 g (33mmol) of 14. The reaction mixture was heated to 95-1000C for 16 hrs at which time TLC showed product together with some Bz-c-CDV. The reaction was allowed to proceed for an additional 8-9 hrs, at which time TLC showed that the reaction had not proceeded much further. At this time, the reaction mixture was cooled to room temperature and the solvent was evaporated under high vacuum. The residue was purified by combiflash using chloroform/MeOH as eluent to furnish 1.8 g of 15 as yellow oil in 42% yield.
[00101] Deprotection and hydrolysis of (15). To 1.7 g (2.56 mmol) of 15 was added 30 mL of concentrated NH4OH and the sealed tube was heated at 950C for 2-3 hrs at which time the solution turned clear. The reaction mixture was then cooled and the TLC showed the reaction to be complete. The NH4OH was evaporated under reduced pressure and the residue was dissolved in 5-10 ml of hot distilled water and dried in a lyophillizer over the weekend. The yellow solids were then washed thoroughly with acetone and the residue was dried in a lyophillizer overnight to furnish 1.6 g of analog 16 as a yellow solid.
Example 5. Preparation of branched methyl esters from the p- toluenesulfonyloxymethyl phosphonates
[00102] Branched methyl esters were prepared from the p- toluenesulfonyloxymethylphosphonates 18 as illustrated in Scheme 4, using synthesis of (S)-HPMPA esters for purposes of illustration. The procedure is based on the method reported by Beadle et ah, J. Med. Chem. 49:2010-2015, 2006.
18
Reagents: a) NaH, N,N-DMF, 500C; b) 80% aq. acetic acid
Scheme 4
[00103] General procedure for preparation of branched methyl alkoxyalkyl p- toluenesulfonyloxymethylphosphonates 18. Diethyl toluenesulfonyloxymethylphosphonate was synthesized from diethyl hydroxymethylphosphonate as described by Holy and Rosenberg, ((1982) Collect. Czech. Chem. Commun. 47:3447-3463). Bromotrimethylsilane (27 g, 175 mmol) was added to a solution of diethyl toluenesulfonyloxymethylphosphonate (9.5 g, 29.5 mmol) in dichloromethane (anhydrous, 150 mL). The mixture was stirred at room temperature under a N2 atmosphere for 18 h. The mixture was then concentrated under vacuum to remove solvent and excess TMSBr, then redissolved in dichloromethane (150 mL) and cooled to O0C with an ice bath. N5N-DMF (0.5 mL) was added, and a solution of oxalyl chloride (22 g, 175 mmol) in CH2Cl2 (50 mL) was added dropwise over 30 min, and then the solution was stirred an additional 5 h. The mixture was evaporated to an oil, which was redissolved in Et2O (100 mL). A solution of the branched methyl alkoxyalkanol 6 (21.5 mmol) and pyridine (10 mL) in Et2O (50 mL) was added, and stirring was continued for about 3 hours or until TLC analysis (1:1 hexanes/ethyl acetate) indicated complete phosphonylation of the alcohol. The reaction mixture was then added to cold saturated NaHCO3 and vigorously stirred one hour. After hydrolysis was complete, the organic layer was separated, dried over MgSO4 and evaporated under vacuum to give the crude esters, which were purified by flash chromatography (15% EtOH/CH2Cl2).
3-(15-methyl hexadecyloxy)propyl p-toluenesulfonyloxymethylphosphonate was prepared using this procedure, MS-ESI (m/z) 561.07 (M + Na)+. [00104] General procedure for preparation of branched methyl esters from the p- toluenesulfonyloxymethylphosphonates 18 is illustrated in Scheme 4 using (S)-HPMPA for purposes of illustration. Briefly, (S)-9-[3-trityloxy-2-hydroxypropyl]-N6-trityl- adenine 17 was prepared from adenine and (S)-trityl glycidyl ether (Daiso Co., Ltd., Japan) following the method of Webb ((1989) Nucleosides & Nucleotides 8:619-624). Sodium hydride (24 mg, 1.0 mmol) was added to a stirred solution of (S)-9-[3-trityloxy- 2-hydroxypropyl]-N6-trityladenine (640 mg, 0.62 mmol) in dry triethylamine (10 mL). After 15 min., the appropriate alkoxyalkyl toluenesulfonyloxymethylphosphonate (0.65 mmol) was added and the reaction mixture was heated to 5O0C and kept overnight. After cooling, the mixture was quenched with brine and extracted with ethyl acetate (3 x 15 mL). The organic extracts were dried over MgSO4 and concentrated under vacuum to provide the fully protected (S)-HPMPA esters. The residue was purified by flash chromatography (10% EtOH/CH2Cl2). For purposes of illustration 3-(15-methyl- hexadecyloxy)propyl (S)-9-[3-trityloxy-2-(phosphonomethoxy)propyl]-N6-trityl- adenine was prepared using this general method.
[00105] Deprotection and isolation of (S)-HPMPA alkoxyalkyl esters 19. Fully protected (S)-HPMPA esters were suspended in 80% aqueous acetic acid (20 mL/mmol) and heated to 6O0C for 1 hour, or until detritylation was complete as determined by TLC analysis. After cooling, the solvent was evaporated and the products 19 were purified by flash chromatography. Elution with 30% MeOH/CH2Cl2 provided 3-(15-methylhexadecyloxy)propyl (S)-HPMPA,, MS-ESI (jn/z) 600.32 (MH)+ as a white solid. Example 6. Preparation of alkenyloxyalkyl esters of acyclic nucleoside phosphonates
[00106] A general method for the synthesis of alkenyloxyalkyl esters having a terminal double bond is outlined in Scheme 5 using the nucleoside phosphonate cidofovir for purposes of illustration.
BnO(CH2)5OH TsCl BnO(CH2)5OTs 20 quant. 21
TsCl
HO OTBDMS quant. HO OTBDMS
22 23
Jr I) Mg
^
2) Li2CuCl4, 23 (CH2)14OTBDMS
24
95% 25
TBAF t ^
(CH2)14OH
77% 26
I) Mg Jr 2) Li2CuCl4, 21 ^ XCH2)J4OBn
24 95% 27
BCl ^ (CH2)J4OH
94% 26
DCM, Et3N, TsCl, DMAP
HO' ^ OTBDMS ^ ocιv TsO' v OTBDMS
28 16 hr* 85% 29
^. NaH, NMP, 29 t ^CH2)i4O(CH2)3OTBDMS
(CH2)J4OH
65% 26 30
75% 31 ^(CH2)14O(CH2)3OH MsCl Et3N 1 ^(CH2)14O(CH2)3OH 31 quant. 32
Scheme 5
[00107] Toluene-4-sulfonic acid-5-benzyloxy-pentyl ester (21). 4.0 g (20 mmol) of commercially available 20 was dissolved in 40 mL of dry DCM. 4.2 mL (30 mmols) of Et3N was added followed by 4.2 g (22 mmols) of TsCl and catalytic DMAP and the reaction was stirred for 16 h. 100 mL of DCM was then added and the reaction mixture was washed successively with saturated aq. NaHCO3 (Ix), water (Ix) and brine (Ix), dried over MgSO4, and the solvent was evaporated under reduced pressure. The crude was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 7.0 g of tosylate 21 in quantitative yield as colorless oil.
[00108] Toluene-4-sulfonic acid-5-tert-butyl-dimethyl-silanyloxy-pentyl ester (23). 5.0 mL (20 mmols) of commercially available 22 was dissolved in 40 mL of dry DCM. 4.2 niL (30 mmols) of Et3N was added followed by 4.2 g (22 mmols) of TsCl and catalytic DMAP and the reaction was stirred for 16 h. 100 rnL of DCM was then added and the reaction mixture was washed successively with saturated aq. NaHCO3 (Ix), water (Ix) and brine (Ix), dried over MgSO4, and the solvent was evaporated under reduced pressure. The crude product was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 7.4 g of target tosylate 23 in quantitative yield as colorless oil.
[00109] Tert-butyl-hexadec-15-enyloxy-dimethylsilane (25). Into a clean flame dried and Ar flushed 500 mL RBF was added 14.2 mL (65 mmol) of commercially available 24. To this was added 100 mL of anhydrous THF, 1.9 g (78 mmol) of Mg turnings and a pellet of I2 to accelerate the reaction and the reaction mixture was refluxed for 2 h. It was then cooled to room temperature and further to -780C. To this was added 7.4 g (20.0 mmol) of 23 in 20 mL of anhydrous THF followed by addition of 6.5 mL (0.65 mmol) of lithium cuprate. The resulting mixture was allowed to warm to room temperature while stirring overnight followed by quenching with saturated aq. NH4Cl and extracted with EtOAc (3x). The combined EtOAc layer was then successively washed with water, saturated aq. NaHCO3 and brine, dried over MgSO4 and the solvents evaporated under reduced pressure. The residue was then purified by combiflash using hexane/EtOAc as eluent to furnish 6.7 g of 25 as colorless oil in 95% yield. [00110] Hexadec-15-en-l-ol (26). To 11.0 g (31 mmol) of 25 was added 124 mL of a 1 M solution of TBAF in THF and the reaction was stirred for 16 h at room temperature. The reaction mixture was then quenched with saturated solution of aq. NH4Cl and THF was evaporated under reduced pressure. The aq. solution was then diluted with 200 mL of water and the target product was extracted with Et2O (3x), the combined organic layers were then washed with brine (Ix), dried over MgSO4 and the solvent was removed under reduced pressure. The crude was then purified by combiflash using hexane/ethyl acetate as eluent to furnish 5.7 g of target product 26 in 77% yield as colorless oil.
[00111] Hexadec- 15-enyloxymethyl-benzene (21). Into a clean flame dried and Ar flushed 500 mL RBF was added 14.2 mL (65 mmol) of commercially available 24. To this mixture was added 100 mL of anhydrous THF, 1.9 g (78 mmol) of Mg turnings and a pellet of I2 to accelerate the reaction. The reaction mixture was refluxed for 2 h and then cooled to room temperature and further to -780C. To this was added 7.0 g (20.0 mmol) of 21 in 20 mL of anhydrous THF followed by addition of 6.5 mL (0.65 mmol) of lithium cuprate. The resulting mixture was allowed to warm to room temperature while stirring overnight followed by quenching with saturated aq. NH4Cl and extracted with EtOAc (3x). The combined EtOAc layers were then successively washed with water, saturated aq. NaHCO3 and brine, dried over MgSO4 and the solvents evaporated under reduced pressure. The residue was then purified by combiflash using hexane/EtOAc as eluent to furnish 6.3 g of the compound 27 as colorless oil in 95% yield.
[00112] Hexadec-15-en-l-ol (26). Into clean flame dried Ar flushed 500 mL RBF was placed 6.3 g (19 mmol) of 27 and 100 mL of dry DCM and the mixture was cooled to - 780C. 95 mL of BCl3 (1.0 M solution in DCM) was then slowly added to the above solution. After complete addition the cooling bath was removed and the reaction mixture was allowed to warm to room temperature. Stirring was continued for an additional 30 minutes at room temperature after which the reaction was cooled in an ice- water bath and quenched very cautiously! ! (as it turns violent) with 100 mL of water. The organic layer was then separated and washed with water, dried over MgSO4 and the solvents evaporated under vacuum. The crude was then purified with combiflash using hexane/ethylacetate as eluent to furnish 4.3 g of 26 in 94% yield. [00113] Toluene-4-sulfonic acid-3-tert-butyl-dimethyl-silanyloxy-propyl ester (29). 25.0 mL (115 mmols) of commercially available 28 was dissolved in 100 mL of dry DCM and 24.2 mL (173 mmols) of Et3N was added and the mixture was cooled to O0C. To this solution was then added 24.2 g (127 mmols) of TsCl and catalytic DMAP. The reaction was stirred for 16 h. 300 mL of DCM was then added and the reaction mixture was washed successively with saturated aq. NaHCO3 (Ix), water (Ix) and brine (Ix), dried over MgSO4, and the solvent was evaporated under reduced pressure. The crude was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 34 g of target tosylate 29 in 85% yield as colorless oil.
[00114] Tert-butyl-3-hexadec- 15-enyloxy-propoxy-dimethylsilane (30). Into a clean flame dried and Ar flushed 2 L RBF was added 12 g (50 mmol) of (26) and 150 mL of anhydrous DMF was added. This solution was then cooled to O0C and 2.6 g (65 mmol) of NaH was added slowly. After complete addition the reaction was stirred at room temperature for 30 minutes and cooled to O0C. 34.5 g (100 mmol) of 29 was then slowly added to the reaction with vigorous stirring. After complete addition the reaction mixture was heated at 8O0C for 2 h at which time TLC showed complete consumption of 26. The reaction was then cooled to room temperature and quenched with dropwise addition of saturated aq. NH4Cl. 200 mL of water was added and the target product was extracted with EtOAc (3x), the combined organic layers were successively washed with water (3x), brine (Ix) and dried over MgSO4. The solvent was evaporated under reduced pressure and the residue was purified by combiflash using hexane/ethyl acetate as an eluent to furnish 13.3 g of 30 as yellow oil in 65% yield.
[00115] 3-Hexadec-15-enyloxy-propanol (31). To 13.1 g (31.8 mmol) of 30 was added 128 mL of a 1 M solution of TBAF in THF and the reaction was stirred for 16 h at room temperature. The reaction mixture was then quenched with saturated solution of aq. NH4Cl and THF was evaporated under reduced pressure. The aq. solution was then diluted with 200 mL of water and the product was extracted with Et2O (3x), the combined organic layers were then washed with brine (Ix), dried over MgSO4 and the solvent was removed under reduced pressure. The crude was then purified by combiflash using hexane/ethyl acetate as eluent to furnish 7.1 g of target product 31 in 75% yield as colorless oil.
[00116] Methanesulfonic acid-3-hex-dec-15-enyloxy-propyl ester (32). To 7.1g (23.7 mmol) of 31 was added 20 mL of DCM and 5.0 mL (36 mmol) of Et3N and the solution was cooled to O0C. To this cooled solution was added dropwise 2.0 mL (26 mmol) of MsCl and a catalytic amount of DMAP. The reaction was stirred at room temperature overnight. The reaction mixture was then diluted with 50 mL of DCM and washed successively with saturated solution of aq. NaHCO3 (Ix), water (Ix) and brine (Ix), dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude product was then purified by combiflash using hexane/ethyl acetate as eluent to furnish 9 g of target product 32 in quantitative yield.
[00117] Substitution of (32) with Bz-c-CDV. 1.7 g (4.6 mmol) of Bz-c-CDV was dissolved in 10 mL of NMP and 1.2 mL (7 mmol) of DIPEA was added to it followed by 9 g (23.9 mmol) of 32. The reaction was heated at 95-1000C for 24 h at which time the TLC showed the product together with some Bz-c-CDV. At this stage the reaction was allowed to proceed for an additional 4 h, but as the TLC did not show much progress, the reaction mixture was cooled to room temperature and the solvent was evaporated under high vacuum. The residue was purified by combiflash using chloroform/MeOH as eluent to furnish 1.33 g of target product 26 as yellow oil in 45% yield.
[00118] Deprotection and hydrolysis of (33). To 1.3 g (2.0 mmol) of 33 was added 30 mL of concentrated NH4OH and the sealed tube was heated at 950C for 2-3 h at which time the solution turns clear. The reaction mixture was then cooled and the TLC showed the reaction to be complete. The NH4OH was evaporated under reduced pressure and the residue was dissolved in 5-10 mL of hot water and dried in lyophilizer over the weekend. The yellow solids were then washed thoroughly with acetone and the residue was dried in lyophilizer for overnight to furnish 1.1 g of analog 34 as a yellow solid.
Example 7. Preparation of penultimate fluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates
[00119] A general procedure for the preparation of penultimate fluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates is illustrated in Scheme 6, below. Fluoroalkoxyalkyl esters of acyclic nucleoside phosphonates such as 3-(15-fluoro- hexadecyloxy)propyl cidofovir 42 (15-F-HDP-CDV) and 3-(15-fluoro- hexadecyloxy)propyl (S)-HPMPA 43 (15-F-HDP-(S)-HPMPA) can be prepared using this process. Briefly, with reference to Scheme 6, commercially available 2- bromopropanoic acid 35 is reduced to the alcohol with borane:THF complex solution to provide 2-bromo-l-propanol 36. Fluorination of 36 is achieved with l,l,2-trifluoro-2- chloroethyldiethylamine, a mild and safe reagent to convert l-hydroxy-2- halogenoalkanes into the corresponding rearranged fluoride 37. Conversion of 37 into a Grignard reagent followed by reaction with 13-bromo-tridecanol in the presence of the catalyst provides 15-fluorohexadecanol 38. Conversion of alcohol 38 into the methanesulfonate derivative, followed by reaction with 1,3-propanediol provides 3-(15- fluorohexadecyloxy)propan-l-ol 39. Reaction of 39 with cyclic cidofovir or cyclic (S)- HPMPA as described generally in Example 2 (step a), provides the cyclic esters (40 and 41, respectively) which can then be converted to the desired compounds (42 and 43, respectively) using the general method set forth in Example 2 (step b).
d → HO(CH2)14CHFCH3 " HO(CH2)3O(CH2)14CHFCH3
38 39
B = cytosin-l-yl, 40 adenin-9-yl, 41
B O
^O^P-O(CH2)3O(CH2)14CHFCH3 i OH
HO"
3-(15-fluorohexadecyloxy)propyl CDV, 42
3-(15-fluorohexadecyloxy)propyl (S)-HPMPA, 43
Reagents: a) BH3, THF; b) fluoroamine; c) Mg/13-bromo-tridecanol, Li2CuCl4; d) methanesulfonyl chloride, pyridine, then 1,3 -propanediol, NaH, N5N-DMF; e) cCDV or cHPMPA, DIAD, PPh3, N5N-DMF; f) 1 M NaOH
Scheme 6
Example 8. Preparation of terminal fluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates
[00120] A general procedure for the preparation of terminal fluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates is illustrated in Scheme 7, below. Fluoroalkoxyalkyl esters of acyclic nucleoside phosphonates such as 3-(16-fluoro- hexadecyloxy)propyl cidofovir (16-F- HDP-CD V) 49 and 3-(16-fluoro- hexadecyloxy)propyl (S)-HPMPA 50 (16-F-HDP-(S)-HPMPA) can be prepared using this process. Briefly, with reference to Scheme 7, the Grignard reagent prepared from l-bromo-4-fluorobutane and magnesium is reacted with 12-bromododecanol 44 to obtain 16-fluorohexadecanol 45. Reaction of 45 with methanesulfonyl chloride followed by reaction with 1,3-propanediol provides 16-fluorohexadecyloxy-l-propanol 46. Coupling of 46 with cyclic cidofovir or cyclic (S)-HPMPA as described generally in Example 2 (step a), provides the cyclic esters (47 and 48, respectively) which can then be converted to the desired compounds (49 and 50, respectively) using the general method set forth in Example 2 (step b).
Br(CH2)12OH — → FCH2(CH2)15OH — F(CH2)16O(CH2)3OH
44 45 46
B = cytosin-1-yl, 47 B = adenin-9-yl, 48
3-(16-fluorohexadecyloxy)propyl CDV, 49
3-(16-fluorohexadecyloxy)propyl (S)-HPMPA, 50
Reagents: a) Mg/l-bromo-4-fluorobutane, Li2CuCl4, THF; b) methanesulfonyl chloride, pyridine; c) 1,3-propanediol, NaH, N5N-DMF; d) cCDV or cHPMPA, DIAD, PPh3, N5N-DMF; e) IM NaOH
Scheme 7
Example 9. Preparation of terminal pentafluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates
[00121] A general procedure for the preparation of terminal pentafluorinated alkoxyalkyl esters of acyclic nucleoside phosphonates is illustrated in Scheme 8.
NH I4,4 58
Scheme 8
[00122] Toluene-4-sulfonic acid 7, 7, 8, 8, 8-pentafluoro octyl ester (52). Into a clean flame dried 250 rnL RBF under current of N2, was added 11.8 g (53.6 mmol) of commercially available 51 which was then dissolved in 100 mL of dry DCM. To this was added 11.2 mL (80.4 mmol) of triethylamine and the flask was cooled in an ice bath. To this was slowly added 11.3g (59 mmol) of TsCl followed by 62 mg (0.50 mmol) of DMAP. The reaction mixture was stirred for 3 h after which it was diluted with 100 mL of DCM and successively washed with saturated solution of NaHCO3, H2O and brine, dried over MgSO4, filtered and evaporated to dryness. The residue was purified using combiflash (120 g silica column) with hexane/ethyl acetate as solvents to furnish 18 g (90%) of the target product 52 as colorless oil.
[00123] 11-(7 J,8,8,8-pentafluoro-octyloxy)-undec-l-ene (54). Into a clean flame dried 1 L RBF under current of N2, was added 100 mL of dry DMF and 20 mL (96 mmols) of commercially available 53. 2.3 g (57.6 mmol) of NaH was added to the reaction and the mixture was stirred for 3 h at room temperature. 18g (48 mmols) of 52 was dissolved in 50 mL of DMF and was slowly added to the above reaction mixture. After complete addition the stirring was continued for 2 h at room temperature and at 800C for 2 h. The reaction was then cooled in an ice bath and quenched with a solution of saturated aq. NH4Cl. 300 mL of water was then added and reaction mixture was extracted with DCM (4x). The combined DCM layers were then washed with H2O (3x), brine (Ix), dried over MgSO4, filtered and the solvent was evaporated under vacuum. The residue was then purified using combiflash (120 g silica column) with hexane/ethyl acetate as eluent to furnish 12.86 g (72%) of the target product 54 as colorless oil.
[00124] 11-(7 ,7,8,8,8-pentafluoro-octyloxy)-undecan-l-ol (55). Into a clean flame dried 1 L RBF under current of N2, was put 12.86g (34.48 mmol) of 54. To this was added 172 mL (86.2 mmol) of 9-BBN (0.5 M in THF). The reaction was stirred for 3 h. 23 mL of 30% H2O2 was then added dropwise to the reaction mixture followed by 49 mL of 15% aq. NaOH and the reaction mixture was stirred at 85°C for 3 h. The mixture was then cooled to room temperature and THF was evaporated and the residue was diluted with H2O and extracted with EtOAc (3x). The combined organic layer was then washed with brine (Ix), dried over MgSO4, filtered and the solvent was evaporated under vacuum. The residue was then purified using combiflash (120 g silica column) with hexane/ethyl acetate as eluent to furnish 10.15 g (76%) of compound 55 as a white solid.
[00125] Methanesulfonic acid 11- (7,7,8,8,8-pentafluoro-octyloxy)-undecyl ester (56). Into a clean flame dried 250 mL RBF under current of N2, was added 10.10 g (26 mmol) of 55. To this was added 100 mL of DCM and 5.5 mL (39 mmol) of Et3N. The reaction mixture was then cooled in an ice-water bath and 2.3 mL (29 mmol) of MsCl was added and stirred for 12 h at room temperature. The reaction was then diluted with 200 niL of DCM and washed successively with sat. aq. NaHCO3 (Ix), H2O (2x), brine (Ix), dried over MgSO4, filtered and the solvent evaporated under vacuum. The residue was then purified using combiflash (12Og silica column) with hexane/ethyl acetate as eluent to furnish 10.15 g (84%) of target product 56 as a white solid. [00126] l l-(7J,8,8,8-pentafluoro-octyloxy)-undecyl N4-benzoyl-cvcric cidofovir (57). Into a clean flame dried 250 mL RBF under current of N2, was placed 2.0 g (5.45 mmol) of benzoyl protected c-CDV followed by 40 mL of dry NMP and the mixture was stirred until the solution turned clear. To this was then added 2.9 mL (16.35 mmol) of DIPEA and 10.15 g (21.7 mmols) of 56 followed by 1.3 g (11 mmol) of NaI. The reaction mixture was then stirred at 900C for 16 h. Solvents were then evaporated under vacuum and the residue was purified using combiflash (12Og silica column) with CHCl3/MeOH as eluent to furnish 1.8 g (45%) of the target product 57 as a yellow solid.
[00127] 11-C7, 7, 8, 8, 8-pentafluoro-octyloxy)-undecyl cidofovir, ammonium salt (58). 1.8 g (2.44 mmol) of 57 was put in a tube and 40 mL of NH4OH was added and the tube was sealed. The reaction mixture was then stirred at 800C in the sealed tube for 12 h. It was then cooled to room temperature after which NH4OH was evaporated under vacuum and the residue was dissolved in 10 mL of H2O and lyophilized. The whitish yellow solid was then washed with acetone (6x) and filtered under suction. The solids were then dried in high vacuum for 16 h to furnish 1.46 g (91%) of the target product 58 as whitish yellow solid in the form of ammonium salt. 1H NMR, 31P NMR, 19F NMR, elemental analysis and LRMS data were all consistent with the structure of the target product 58.
Example 10. Evaluation of antiviral activity of penultimate branched methyl alkoxyalkyl analogs of cidofovir (CDV) and 9-(S)-(3-hydroxy-2- phosphonomethoxypropyP-adenine ((S)-HPMPA) against vaccinia virus and cowpox virus in vitro
[00128] Virus pool preparation. The vaccinia virus strain, Copenhagen, and cowpox virus, strain Brighton, stock pools were obtained from John Huggins of the U.S. Army Medical Research Institute for Infectious Diseases, Frederick, Md. These pools were prepared in Vero cells and were diluted 1:50 to provide working stocks. [00129] Plaque reduction assay for efficacy. Two days prior to use, HFF cells were plated on six-well plates and incubated at 37°C with 10% CO2 and 90% humidity. On the day of the assay, the drugs were made up at twice the desired concentration in 2x minimal essential medium (MEM) containing 5% fetal bovine serum (FBS) and antibiotics and diluted serially 1:5 in 2x MEM to provide six concentrations of drug. The initial starting concentration was usually 200 μM and ranged down to 0.06 μM. The virus to be used was diluted in MEM containing 10% FBS to a desired concentration which would give 20 to 30 plaques per well. The medium was then aspirated from the wells, and 0.2 mL of virus was added to each well in triplicate, with 0.2 mL of medium being added to drug toxicity wells. The plates were incubated for 1 h with shaking every 15 min. After the incubation period, an equal amount of 1% agarose was added to an equal volume of each drug dilution. This gave final drug concentrations beginning with 100 μM and ending with 0.03 μM and a final agarose overlay concentration of 0.5%. The drug-agarose mixture was added to each well in 2 mL volumes, and the plates were incubated for 3 days, after which the cells were stained with a 1.5% solution of neutral red. After a 5- to 6-h incubation period, the stain was aspirated and the plaques were counted using a stereomicroscope at xlO magnification. The MacSynergy II, version 1, computer program was used to calculate the 50% effective concentration (EC50). The results are set forth in Tables 3 and 4. [00130] Neutral-red uptake assay for toxicity. Twenty-four hours prior to the assay, HFF cells were plated on 96- well plates at a concentration of 2.5 x 10 per mL. After 24 h, the medium was aspirated and 125 μL of drug was added to the first row of wells and then diluted serially 1:5 using the Beckman BioMek liquid-handling system. After the addition of the drug, the plates were incubated for 7 days in a CO2 incubator at 37°C. At that time, the medium with drug was aspirated, and 200 μl of 0.01% neutral red in phosphate-buffered saline (PBS)/well was added and incubated for 1 h. The dye was aspirated, and the cells were washed with PBS using a Nunc plate washer. After the PBS was removed, 200 μL of 50% ethanol-1% glacial acetic acid (in H20)/well was added. The plates were placed on a rotary shaker for 15 min, and the optical densities were read at 540 nm on a Bio-tek plate reader. The results are set forth in Tables 3 and 4. Table 3. Activity of penultimate branched methyl alkoxyalkyl analogs of CDV against vaccinia virus and cowpox virus in HFF cells
3SI (Selectivity Index) = CC50ZEC5O
Abbreviations: CDV, cidofovir; HDP-CDV, hexadecyloxypropyl-CDV; 17M-ODE-CDV, 17-methyl- octadecyloxyethyl cidofovir; 15M-HDP-CDV, 15-methyl-hexadecyloxypropyl cidofovir; 14M-PDP- CDV, 14-methyl-pentadecyloxypropyl cidofovir; 13M-TDP-CDV, 13-methyl-tetradecyloxypropyl cidofovir; 12M-TrDP-CDV, 12-methyl-tridecyloxypropyl cidofovir
Table 4. Activity of penultimate branched methyl alkoxyalkyl analogs of (S)- HPMPA against vaccinia virus and cowpox virus in HFF cells
3SI (Selectivity Index) = CC50/EC50 Abbreviations: CDV, cidofovir; (S)-HPMPA, 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)-adenine; HDP-, hexadecyloxypropyl; ODE-, octadecyloxypropyl; 15M-HDP-, 15-methyl-hexadecyloxypropyl-; 17M-ODE-, 17-methyl-octadecyloxyethyl- Example 11. Evaluation of antiviral activity of penultimate branched methyl alkoxyalkyl analogs of CDV and (S)-HPMPA against ectromelia virus in vitro
[00131] Cells and Viruses. BS-C-I cells (ATCC CCL 26) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine serum fetal clone III (Hyclone, Logan, UT), 2 mM L-glutamine (GIBCO, Grand Island, NY), 100 U/mL penicillin (GIBCO), and 100 μg/mL streptomycin (GIBCO). A plaque-purified isolate of the MOS strain of ECTV (ATCC VR-1374) designated MOS-3-P2 was propagated in an African green monkey kidney cell line, BS-C-I. Virus was purified through a sucrose cushion. Virus suspensions were serially diluted in PBS + 1% sera, absorbed to monolayers for 1 h at 37°C, and overlayed with a suspension of 1% carboxyl methyl cellulose in DMEM +5% Fetal clone III. After 4 days at 37°C, virus plaques were visualized and virus inactivated by the addition to each well of 0.5 mL of a 0.3% crystal violet/10% formalin solution.
[00132] Plaque reduction assay. CV-I cells were plated in wells of a 24- well cluster plate. Each monolayer was infected with 75 plaque forming units (PFU) of indicator virus in 0.1 mL of DMEM +5% Fetal clone III for 60 min at 370C. Media was removed by aspiration and standard virus overlay media containing no drug or the test drug at concentrations ranging from 0.05 to 50 μM was added. The plates were incubated at 37°C for 3-4 days for ECTV and 2 days for VACV-WR, monolayers were stained, and plaques counted using a stereomicroscope. The EC50 concentration for each drug was calculated. The results are set forth in Table 5.
Table 5. Activity of penultimate branched methyl alkoxyalkyl analo s of CDV and (S)-HPMPA a ainst ectromelia infection in vitro
Abbreviations as set forth in Tables 3 and 4
Example 12. Activity of oral 15M-HDP-(S)-HPMPA in lethal ectromelia virus infection
[00133] In vivo drug evaluation: A/Ncr mice were injected to effect with an anesthetic cocktail (ketamine 90 mg/kg / xylazine 10 mg/kg), held at 45° C from the vertical on a intubation platform, and inoculated with 5μl of virus suspension in each naris for a total challenge dose of 140 PFU (-280 xLD50). Approximately 2.5 min following inoculation of virus, the mice were returned to their cage. Four hours following exposure to ECTV, groups of mice were treated by gavage with 0.1 ml of sterile, distilled water alone or water containing the test compound. This treatment was repeated on days 1, 2, 3, and 4 for a total of five doses. The mice were observed over 21 days for clinical signs of disease (morbidity) and mortality. The results are set forth in Table 6.
Table 6. Activity of oral HDP-(S)-HPMPA, HDP-CDV and penultimate branched methyl analogs against lethal ectromelia infection in vivo
Abbreviations: HDP, hexadexyloxypropyl; 15M-HDP, 15-methyl-hexadecyloxypropyl; 13M-TDP, 13-methyl-tetradecyloxypropyl; 14M-PDP, 14-methyl-pentadecyloxypropyl. Drug administered orally 4 hrs after infection daily for 5 days. MDD, Mean day of death
Example 13. Metabolic stability testing with liver S9 fractions
[00134] Target compounds were incubated in pooled S9 liver fractions from monkey and human liver (purchased from a commercial source) at 370C. The control sample (immediately quenched) was used to determine the response at time zero. The ratio between the response of the incubated samples and time zero indicated the % parent compound remaining. Compounds were dissolved in DMSO and serially diluted with buffer to a concentration suitable for the assay (1 to 10 μM). A portion of a high concentration dilution (-500 mM) was used to determine the LC-MS/MS conditions for analysis (ionization polarity, SRM transition, collision energy). 7-ethoxycoumarin was included as a control. Negative controls (without S9) were included to check the stability of the test compounds at incubation conditions. All assays were performed in triplicate and the % parent compound remaining was reported. Incubations were generally performed in microtiter plates with a protein concentration of 3 mg/mL and a compound concentration of 1 μM. Reactions were sampled at the specified time points and stopped by the addition of a cold acetonitrile/water solution. The quenched plates were centrifuged and subsequently analyzed by fast gradient LC-MS/MS. The results are set forth in Figure 3.

Claims

1. A nucleoside phosphonate selected from the group of compounds having the following structures:
wherein
R is selected from the group consisting of -Ri-O-R2, wherein Ri is selected from the group consisting of an optionally substituted Ci to Cn alkyl group and R2 is selected from the group consisting of a C6 to Cn alkyl group or a C6 to Cn alkenyl group; wherein said Ce to C17 alkyl group is substituted with one or more alkyl groups selected from the group consisting of methyl, ethyl, propyl, or cycloalkyl, including cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said C6 to Ci7 alkyl group includes one or more substituents at or near the terminal position of the alkyl group; and wherein said Ce to Ci7 alkenyl group is optionally-substituted with an alkyl group selected from the group consisting of methyl, ethyl, propyl, a cycloalkyl group including, cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein the said Ce to Ci7 alkenyl group contains one or more double bonds, including a terminal double bond;
B is selected from a purine or pyrimidine base; and
A is a counterion selected from the group consisting of H+, Li+, Na+, K+, NH4 +, tetraalkyl ammonium and other tertiary amine salts including triethylamine.
2. The phosphonate of claim 1 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17.
3. The phosphonate of claim 1 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17.
4. The phosphonate of claim 1 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17 and X is a halogen.
5. The phosphonate of claim 4 wherein X is F.
6. The phosphonate of claim 1 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17 and X is independently selected from a halogen.
7. The phosphonate of claim 6 wherein X is F.
8. The phosphonate of claim 6 wherein R is selected from the group of compounds having the following structures:
CH3 CH3 CH3 CH3
HO O
F HO ^^ O
9. The nucleoside phosphonate of claim 1 wherein R is selected to prevent or decrease metabolic degradation of the phosphonate.
10. The nucleoside phosphonate of claim 1 wherein said phosphonate is selected from a compound having the following structures: cyclic cidofovir
11. The nucleoside phosphonate of claim 1 wherein said phosphonate is selected from a compound having the following structures:
cyclic (S)-HPMPA (S)-HPMPA
12. A nucleoside phosphonate selected from the group consisting of: 3-(12- methyltridecyloxy)propyl cyclic cidofovir, 3-(13-methyltetradecyloxy)propyl cyclic cidofovir, 3-(14-methylpentadecyloxy)propyl cyclic cidofovir, 2-(17- methyloctadecyloxy)ethyl cyclic cidofovir, 3-(15-methylhexadecyloxy)propyl cyclic cidofovir, 3-(15-methylhexadecyloxy)ethyl (S)-cyclic HPMPA, 3-(15- methylhexadecyloxy)propyl (S)-cyclic HPMPA, 2-(17-methyloctadecyloxy)ethyl-(S)- cyclic HPMPA, 3-(12-methyltridecyloxy)propyl cidofovir, 3-(13- methyltetradecyloxy)propyl cidofovir, 3-(14-methylpentadecyloxy)propyl cidofovir, 3- (15-methylhexadecyloxy)propyl cidofovir, sodium, 3-(15-methylhexadecyloxy)propyl cidofovir, ammonium,2-(17-methyloctadecyloxy)ethyl cidofovir, 2-(15- methylhexadecyloxy)ethyl cidofovir, 3-(phytanyloxy)propyl cidofovir, 3-(15- methylhexadceyloxy)ethyl-(S)-HPMPA, 2-(17-methyloctadecyloxy)ethyl-(S)-HPMPA, 3-(hex-dec-15-enyloxy)propyl cidofovir, ammonium, 3-(15-fluorohexadecyloxy)propyl cidofovir, 3-(15-fhiorohexadecyloxy)propyl cyclic cidofovir, 3-(15- fluorohexadceyloxy)propyl-(S)-HPMPA, 3-(15-fluorohexadceyloxy)propyl-(S)-cyclic HPMPA, 3-(16-fhiorohexadecyloxy)propyl cidofovir, 3-(16-fluorohexadecyloxy)propyl cyclic cidofovir, 3-(16-fluorohexadceyloxy)propyl-(S)-HPMPA, 3-(16- fluorohexadceyloxy)propyl-(S)-cyclic HPMPA, and l l-(7, 7, 8, 8, 8-pentafluoro- octyloxy)-undecyl cidofovir, ammonium salt.
13. The nucleoside phosphonate of claim 1 wherein said phosphonate is selected from an antiviral or an antineoplastic agent.
14. The nucleoside phosphonate of claim 13, wherein said antiviral agent is a derivative is adefovir, cidofovir, cyclic cidofovir, tenofovir, phosphonomethyoxyethylguanine or HPMPA.
15. A pharmaceutical composition comprising a nucleoside phosphonate according to claim 1 and a pharmaceutically acceptable carrier.
16. A method for treating a viral infection, said method comprising administering to a host in need thereof an effective amount of a phosphonate compound according to claim 1.
17. A nucleoside phosphate selected from the group of compounds having the following structures:
adenine beta-L -nucleosides wherein
R is selected from the group consisting of -Ri-O-R2, wherein Ri is selected from the group consisting of an optionally substituted Ci to Cn alkyl group and R2 is selected from the group consisting of a Ce to Cn alkyl group or a Ce to Cn alkenyl group; wherein said Ce to Cn alkyl group is substituted with one or more alkyl groups selected from the group consisting of methyl, ethyl, propyl, or cycloalkyl, including cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said Ce to Cn alkyl group includes one or more substituents at or near the terminal position of the alkyl group; and wherein said Ce to Cn alkenyl group is optionally-substituted with an alkyl group selected from the group consisting of methyl, ethyl, propyl, a cycloalkyl group including, cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein the said Ce to Cn alkenyl group contains one or more double bonds, including a terminal double bond; B is selected from a purine or pyrimidine base; and A is a counterion selected from the group consisting of H+, Li+, Na+, K+, NH4 +, tetraalkyl ammonium and other tertiary amine salts including triethylamine.
18. The phosphate of claim 17 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17.
19. The phosphate of claim 17 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17.
20. The phosphate of claim 17 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17 and X is a halogen.
21. The phosphate of claim 20 wherein X is F.
22. The phosphate of claim 17 wherein R is selected from the group of compounds having the following structure:
wherein p is selected from 1 to 11 and q is selected from 6 to 17 and X is independently selected from a halogen.
23. The phosphate of claim 22 wherein X is F.
24. The phosphate of claim 17 wherein R is selected from the group of compounds having the following structures:
CH3 HO O
CH3 CH3 CH3 CH3
HO O
F
HO ^^ O
25. The nucleoside phosphate of claim 17 wherein R is selected to prevent or decrease metabolic degradation of the phosphonate.
26 The nucleoside phosphate of claim 17 wherein said phosphate is selected from an antiviral or an antineoplastic agent.
27. The nucleoside phosphate of claim 26, wherein said antiviral agent is a derivative of a compound selected from the group consisting of acyclovir, ganciclovir, AZT, ddl, ddA, d4T, ddC, 3TC, FTC, 2'-C-methyl adenosine, 2'-C-methyl guanosine, 7- deaza-2'-methyl adenosine, 2'-C-methyl cytosine, DAPD, L-FMAU, entecavir, telbivudine and various β-L-2'-deoxycytidine, β-L-2'-deoxyadenine and β-L-2'- deoxythymidine.
28. The nucleoside phosphate of claim 26 wherein said antineoplastic agent is selected from the group consisting of 2/-deoxy-2',2/-difluorocytidine (gemcitibine), (E)- 2'-deoxy-2'-fluoromethylene-cytidine (FMdC), or l-(2-deoxy-2-fluoro-4-thio-β-D- arabinosyl)cytosine (4'-thio-FAC), Ara-C, Ara-G, 5-fluorouridine, 5-fluoro- deoxyuridine, (R)-deoxycoformycin, and fludarabine.
29. A pharmaceutical composition comprising a nucleoside phosphate according to claim 17 and a pharmaceutically acceptable carrier.
30. A method for treating a viral infection, said method comprising administering to a host in need thereof an effective amount of a phosphate compound according to claim 17.
31. A method for treating a growing neoplasm, said method comprising administering to a host in need thereof an effective amount of a phosphate compound according to claim 17.
32. A method for modulating cell proliferation, said method comprising administering to a host in need thereof an effective amount of a phosphate compound according to claim 17.
33. The phosphate ester of the antiviral compound, zanamivir having the following structure: wherein
Z is selected from the group consisting of -P(O)(OR)(OA); wherein
R is selected from the group consisting of -Ri-O-R2, wherein Ri is selected from the group consisting of an optionally substituted Ci to Cn alkyl group and R2 is selected from the group consisting of a Ce to Cn alkyl group or a Ce to Cn alkenyl group; wherein said Ce to Cn alkyl group is substituted with one or more alkyl groups selected from the group consisting of methyl, ethyl, propyl, or cycloalkyl, including cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein said Ce to Cn alkyl group includes one or more substituents at or near the terminal position of the alkyl group; and wherein said Ce to Cn alkenyl group is optionally-substituted with an alkyl group selected from the group consisting of methyl, ethyl, propyl, a cycloalkyl group including, cyclopropyl and/or one or more halogens selected from the group consisting of F, Cl, Br and I; and further wherein the said Ce to Cn alkenyl group contains one or more double bonds, including a terminal double bond; and wherein
A is a counterion selected from the group consisting of H+, Li+, Na+, K+, NH4 +, tetraalkyl ammonium and other tertiary amine salts including triethylamine.
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US7994143B2 (en) 2011-08-09
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US20100249056A1 (en) 2010-09-30
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